Methods and compositions for selecting siRNA of improved functionality

ABSTRACT

Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs by rational design, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing genes. Methods, compositions, and kits generated through rational design of siRNAs are disclosed.

REFERENCE TO TABLES SUBMITTED IN ELECTRONIC FORM

In accordance with PCT Administrative Instructions Part 8, Applicantsubmits a compact disc of tables related to sequences and herebyincorporates by reference the material submitted herewith, on thecompact disk labeled COPY 1—TABLES PART DISK 1/1, TABLES XII and XIII(provided in triplicate, which copies are identical), in files entitledtable-xii.txt, date of creation 26 Apr. 2004, with a size of 110,486 kb,and table-xiii.txt, date of creation 26 Apr. 2004, with a size of 23,146kb; and in accordance with PCT Administrative Instructions Section801(a)(i) on the compact disk labeled CRF (with three further copies,which copies are identical) in a file entitled 13608PCT.txt, date ofcreation 26 Apr. 2004, with a size of 556,776 kb.

FIELD OF INVENTION

The present invention relates to RNA interference (“RNAi”).

BACKGROUND OF THE INVENTION

Relatively recently, researchers observed that double stranded RNA(“dsRNA”) could be used to inhibit protein expression. This ability tosilence a gene has broad potential for treating human diseases, and manyresearchers and commercial entities are currently investing considerableresources in developing therapies based on this technology.

Double stranded RNA induced gene silencing can occur on at least threedifferent levels: (i) transcription inactivation, which refers to RNAguided DNA or histone methylation; (ii) siRNA induced mRNA degradation;and (iii) mRNA induced transcriptional attenuation.

It is generally considered that the major mechanism of RNA inducedsilencing (RNA interference, or RNAi) in mammalian cells is mRNAdegradation. Initial attempts to use RNAi in mammalian cells focused onthe use of long strands of dsRNA. However, these attempts to induce RNAimet with limited success, due in part to the induction of the interferonresponse, which results in a general, as opposed to a target-specific,inhibition of protein synthesis. Thus, long dsRNA is not a viable optionfor RNAi in mammalian systems.

More recently it has been shown that when short (18-30 bp) RNA duplexesare introduced into mammalian cells in culture, sequence-specificinhibition of target mRNA can be realized without inducing an interferonresponse. Certain of these short dsRNAs, referred to as small inhibitoryRNAs (“siRNAs”), can act catalytically at sub-molar concentrations tocleave greater than 95% of the target mRNA in the cell. A description ofthe mechanisms for siRNA activity, as well as some of its applicationsare described in Provost et al., Ribonuclease Activity and RNA Bindingof Recombinant Human Dicer, E.M.B.O. J., 2002 Nov. 1; 21(21): 5864-5874;Tabara et al., The dsRNA Binding Protein RDE-4 Interacts with RDE-1,DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 2002,Jun. 28;109(7):861-71; Ketting et al., Dicer Functions in RNAInterference and in Synthesis of Small RNA Involved in DevelopmentalTiming in C. elegans; Martinez et al., Single-Stranded Antisense siRNAsGuide Target RNA Cleavage in RNAi, Cell 2002, Sep. 6; 110(5):563;Hutvagner & Zamore, A micro RNA in a multiple-turnover RNAi enzymecomplex, Science 2002, 297:2056.

From a mechanistic perspective, introduction of long double stranded RNAinto plants and invertebrate cells is broken down into siRNA by a TypeIII endonuclease known as Dicer. Sharp, RNA interference—2001, GenesDev. 2001, 15:485. Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs. Bernstein, Caudy, Hammond, & Hannon, Role for abidentate ribonuclease in the initiation step of RNA interference,Nature 2001, 409:363. The siRNAs are then incorporated into anRNA-induced silencing complex (RISC) where one or more helicases unwindthe siRNA duplex, enabling the complementary antisense strand to guidetarget recognition. Nykanen, Haley, & Zamore, ATP requirements and smallinterfering RNA structure in the RNA interference pathway, Cell 2001,107:309. Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleaves the target to induce silencing.Elbashir, Lendeckel, & Tuschl, RNA interference is mediated by 21- and22-nucleotide RNAs, Genes Dev 2001, 15:188, FIG. 1.

The interference effect can be long lasting and may be detectable aftermany cell divisions. Moreover, RNAi exhibits sequence specificity.Kisielow, M. et al. (2002) Isoform-specific knockdown and expression ofadaptor protein ShcA using small interfering RNA, J. of Biochemistry363: 1-5. Thus, the RNAi machinery can specifically knock down one typeof transcript, while not affecting closely related mRNA. Theseproperties make siRNA a potentially valuable tool for inhibiting geneexpression and studying gene function and drug target validation.Moreover, siRNAs are potentially useful as therapeutic agents against:(1) diseases that are caused by over-expression or misexpression ofgenes; and (2) diseases brought about by expression of genes thatcontain mutations.

Successful siRNA-dependent gene silencing depends on a number offactors. One of the most contentious issues in RNAi is the question ofthe necessity of siRNA design, i.e., considering the sequence of thesiRNA used. Early work in C. elegans and plants circumvented the issueof design by introducing long dsRNA (see, for instance, Fire, A. et al.(1998) Nature 391:806-811). In this primitive organism, long dsRNAmolecules are cleaved into siRNA by Dicer, thus generating a diversepopulation of duplexes that can potentially cover the entire transcript.While some fraction of these molecules are non-functional (i.e., inducelittle or no silencing) one or more have the potential to be highlyfunctional, thereby silencing the gene of interest and alleviating theneed for siRNA design. Unfortunately, due to the interferon response,this same approach is unavailable for mammalian systems. While thiseffect can be circumvented by bypassing the Dicer cleavage step anddirectly introducing siRNA, this tactic carries with it the risk thatthe chosen siRNA sequence may be non-functional or semi-functional.

A number of researches have expressed the view that siRNA design is nota crucial element of RNAi. On the other hand, others in the field havebegun to explore the possibility that RNAi can be made more efficient bypaying attention to the design of the siRNA. Unfortunately, none of thereported methods have provided a satisfactory scheme for reliablyselecting siRNA with acceptable levels of functionality. Accordingly,there is a need to develop rational criteria by which to select siRNAwith an acceptable level of functionality, and to identify siRNA thathave this improved level of functionality, as well as to identify siRNAsthat are hyperfunctional.

SUMMARY OF THE INVENTION

The present invention is directed to increasing the efficiency of RNAi,particularly in mammalian systems. Accordingly, the present inventionprovides kits, siRNAs and methods for increasing siRNA efficacy.

According to a first embodiment, the present invention provides a kitfor gene silencing, wherein said kit is comprised of a pool of at leasttwo siRNA duplexes, each of which is comprised of a sequence that iscomplementary to a portion of the sequence of one or more targetmessenger RNA, and each of which is selected using non-target specificcriteria.

According to a second embodiment, the present invention provides amethod for selecting an siRNA, said method comprising applying selectioncriteria to a set of potential siRNA that comprise 18-30 base pairs,wherein said selection criteria are non-target specific criteria, andsaid set comprises at least two siRNAs and each of said at least twosiRNAs contains a sequence that is at least substantially complementaryto a target gene; and determining the relative functionality of the atleast two siRNAs.

In one embodiment, the present invention also provides a method whereinsaid selection criteria are embodied in a formula comprising:(−14)*G₁₃−13*A₁−12*U₇−11*U₂−10*A₁₁−10*U₄−10*C₃−10*C₅−10*C₆−9*A₁₀−9*U₉−9*C₁₈−8*G₁₀−7*U₁−7*U₁₆−7*C₁₇−7*C₁₉+7*U₁₇+8*A₂+8*A₄+8*A₅+8*C₄+9*G₈+10*A₇+10*U₁₈+11*A₁₉+11*C₉+15*G₁+18*A₃+19*U₁₀−Tm−3*(GC_(total))−6*(GC₁₅₋₁₉)−30*X; or   Formula VIII(−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(numberof A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula Xwherein position numbering begins at the 5′-most position of a sensestrand, and

-   -   A₁=1 if A is the base at position 1 of the sense strand,        otherwise its value is 0;    -   A₂=1 if A is the base at position 2 of the sense strand,        otherwise its value is 0;    -   A₃=1 if A is the base at position 3 of the sense strand,        otherwise its value is 0;    -   A₄=1 if A is the base at position 4 of the sense strand,        otherwise its value is 0;    -   A₅=1 if A is the base at position 5 of the sense strand,        otherwise its value is 0;    -   A₆=1 if A is the base at position 6 of the sense strand,        otherwise its value is 0;    -   A₇=1 if A is the base at position 7 of the sense strand,        otherwise its value is 0;    -   A₁₀=1 if A is the base at position 10 of the sense strand,        otherwise its value is 0;    -   A₁₁=1 if A is the base at position 11 of the sense strand,        otherwise its value is 0;    -   A₁₃=1 if A is the base at position 13 of the sense strand,        otherwise its value is 0;    -   A₁₉=1 if A is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   C₃=1 if C is the base at position 3 of the sense strand,        otherwise its value is 0;    -   C₄=1 if C is the base at position 4 of the sense strand,        otherwise its value is 0;    -   C₅=1 if C is the base at position 5 of the sense strand,        otherwise its value is 0;    -   C₆=1 if C is the base at position 6 of the sense strand,        otherwise its value is 0;    -   C₇=1 if C is the base at position 7 of the sense strand,        otherwise its value is 0;    -   C₉=1 if C is the base at position 9 of the sense strand,        otherwise its value is 0;    -   C₁₇=1 if C is the base at position 17 of the sense strand,        otherwise its value is 0;    -   C₁₈=1 if C is the base at position 18 of the sense strand,        otherwise its value is 0;    -   C₁₉=1 if C is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   G₁=1 if G is the base at position 1 on the sense strand,        otherwise its value is 0;    -   G₂=1 if G is the base at position 2 of the sense strand,        otherwise its value is 0;    -   G₈=1 if G is the base at position 8 on the sense strand,        otherwise its value is 0;    -   G₁₀=1 if G is the base at position 10 on the sense strand,        otherwise its value is 0;    -   G₁₃=1 if G is the base at position 13 on the sense strand,        otherwise its value is 0;    -   G₁₉=1 if G is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   U₁=1 if U is the base at position 1 on the sense strand,        otherwise its value is 0;    -   U₂=1 if U is the base at position 2 on the sense strand,        otherwise its value is 0;    -   U₃=1 if U is the base at position 3 on the sense strand,        otherwise its value is 0;    -   U₄=1 if U is the base at position 4 on the sense strand,        otherwise its value is 0;    -   U₇=1 if U is the base at position 7 on the sense strand,        otherwise its value is 0;    -   U₉=1 if U is the base at position 9 on the sense strand,        otherwise its value is 0;    -   U₁₀=1 if U is the base at position 10 on the sense strand,        otherwise its value is 0;    -   U₁₅=1 if U is the base at position 15 on the sense strand,        otherwise its value is 0;    -   U₁₆=1 if U is the base at position 16 on the sense strand,        otherwise its value is 0;    -   U₁₇=1 if U is the base at position 17 on the sense strand,        otherwise its value is 0;    -   U₁₈=1 if U is the base at position 18 on the sense strand,        otherwise its value is 0.    -   GC₁₅₋₁₉=the number of G and C bases within positions 15-19 of        the sense strand, or within positions 15-18 if the sense strand        is only 18 base pairs in length;    -   GC_(total)=the number of G and C bases in the sense strand;    -   Tm=100 if the siRNA oligo has the internal repeat longer then 4        base pairs, otherwise its value is 0; and    -   X=the number of times that the same nucleotide repeats four or        more times in a row.

According to a third embodiment, the invention provides a method fordeveloping an algorithm for selecting siRNA, said method comprising: (a)selecting a set of siRNA; (b) measuring gene silencing ability of eachsiRNA from said set; (c) determining relative functionality of eachsiRNA; (d) determining improved functionality by the presence or absenceof at least one variable selected from the group consisting of thepresence or absence of a particular nucleotide at a particular position,the total number of As and Us in positions 15-19, the number of timesthat the same nucleotide repeats within a given sequence, and the totalnumber of Gs and Cs; and (e) developing an algorithm using theinformation of step (d).

According to a fourth embodiment, the present invention provides a kit,wherein said kit is comprised of at least two siRNAs, wherein said atleast two siRNAs comprise a first optimized siRNA and a second optimizedsiRNA, wherein said first optimized siRNA and said second optimizedsiRNA are optimized according a formula comprising Formula X.

According to a fifth embodiment, the present invention provides a methodfor identifying a hyperfunctional siRNA, comprising applying selectioncriteria to a set of potential siRNA that comprise 18-30 base pairs,wherein said selection criteria are non-target specific criteria, andsaid set comprises at least two siRNAs and each of said at least twosiRNAs contains a sequence that is at least substantially complementaryto a target gene; determining the relative functionality of the at leasttwo siRNAs and assigning each of the at least two siRNAs a functionalityscore; and selecting siRNAs from the at least two siRNAs that have afunctionality score that reflects greater than 80 percent silencing at aconcentration in the picomolar range, wherein said greater than 80percent silencing endures for greater than 120 hours.

According to a sixth embodiment, the present invention provides ahyperfunctional siRNA that is capable of silencing Bc12.

According to a seventh embodiment, the present invention provides amethod for developing an siRNA algorithm for selecting functional andhyperfunctional siRNAs for a given sequence. The method comprises:

-   -   (a) selecting a set of siRNAs;    -   (b) measuring the gene silencing ability of each siRNA from said        set;    -   (c) determining the relative functionality of each siRNA;    -   (d) determining the amount of improved functionality by the        presence or absence of at least one variable selected from the        group consisting of the total GC content, melting temperature of        the siRNA, GC content at positions 15-19, the presence or        absence of a particular nucleotide at a particular position,        relative thermodynamic stability at particular positions in a        duplex, and the number of times that the same nucleotide repeats        within a given sequence; and    -   (e) developing an algorithm using the information of step (d).        According to this embodiment, preferably the set of siRNAs        comprises at least 90 siRNAs from at least one gene, more        preferably at least 180 siRNAs from at least two different        genes, and most preferably at least 270 and 360 siRNAs from at        least three and four different genes, respectively.        Additionally, in step (d) the determination is made with        preferably at least two, more preferably at least three, even        more preferably at least four, and most preferably all of the        variables. The resulting algorithm is not target sequence        specific.

In another embodiment, the present invention provides rationallydesigned siRNAs identified using the formulas above.

In yet another embodiment, the present invention is directed tohyperfunctional siRNA.

The ability to use the above algorithms, which are not sequence orspecies specific, allows for the cost-effective selection of optimizedsiRNAs for specific target sequences. Accordingly, there will be bothgreater efficiency and reliability in the use of siRNA technologies.

For a better understanding of the present invention together with otherand further advantages and embodiments, reference is made to thefollowing description taken in conjunction with the examples, the scopeof which is set forth in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a model for siRNA-RISC interactions. RISC has the abilityto interact with either end of the siRNA or miRNA molecule. Followingbinding, the duplex is unwound, and the relevant target is identified,cleaved, and released.

FIG. 2 is a representation of the functionality of two hundred andseventy siRNA duplexes that were generated to target human cyclophilin,human diazepam-binding inhibitor (DB), and firefly luciferase.

FIG. 3 a is a representation of the silencing effect of 30 siRNAs inthree different cells lines, HEK293, DU145, and Hela. FIG. 3 b shows thefrequency of different functional groups (>95% silencing (black), >80%silencing (gray), >50% silencing (dark gray), and <50% silencing(white)) based on GC content. In cases where a given bar is absent froma particular GC percentage, no siRNA were identified for that particulargroup. FIG. 3 c shows the frequency of different functional groups basedon melting temperature (Tm).

FIG. 4 is a representation of a statistical analysis that revealedcorrelations between silencing and five sequence-related properties ofsiRNA: (A) an A at position 19 of the sense strand, (B) an A at position3 of the sense strand, (C) a U at position 10 of the sense strand, (D) abase other than G at position 13 of the sense strand, and (E) a baseother than C at position 19 of the sense strand. All variables werecorrelated with siRNA silencing of firefly luciferase and humancyclophilin. siRNAs satisfying the criterion are grouped on the left(Selected) while those that do not, are grouped on the right(Eliminated). Y-axis is “% Silencing of Control.” Each position on theX-axis represents a unique siRNA.

FIGS. 5A and 5B are representations of firefly luciferase andcyclophilin siRNA panels sorted according to functionality and predictedvalues using Formula VIII. The siRNA found within the circle representthose that have Formula VIII values (SMARTscores™) above zero. siRNAoutside the indicated area have calculated Formula VIII values that arebelow zero. Y-axis is “Expression (% Control).” Each position on theX-axis represents a unique siRNA.

FIG. 6A is a representation of the average internal stability profile(AISP) derived from 270 siRNAs taken from three separate genes(cyclophilin B, DBI and firefly luciferase). Graphs represent AISPvalues of highly functional, functional, and non-functional siRNA. FIG.6B is a comparison between the AISP of naturally derived GFP siRNA(filled squares) and the AISP of siRNA from cyclophilin B, DBI, andluciferase having >90% silencing properties (no fill) for the antisensestrand. “DG” is the symbol for ΔG, free energy.

FIG. 7 is a histogram showing the differences in duplex functionalityupon introduction of basepair mismatches. The X-axis shows the mismatchintroduced in the siRNA and the position it is introduced (e.g., 8C>Areveals that position 8 (which normally has a C) has been changed to anA). The Y-axis is “% Silencing (Normalized to Control).”

FIG. 8 a is histogram that shows the effects of 5′ sense and antisensestrand modification with 2′-O-methylation on functionality. FIG. 8 b isan expression profile showing a comparison of sense strand off-targeteffects for IGF1R-3 and 2′-O-methyl IGF1R-3. Sense strand off-targets(lower box) are not induced when the 5′ end of the sense strand ismodified with 2′-O-methyl groups (top box).

FIG. 9 shows a graph of SMARTscores™ versus RNAi silencing values formore than 360 siRNA directed against 30 different genes. siRNA to theright of the vertical bar represent those siRNA that have desirableSMARTscores™.

FIGS. 10A-E compare the RNAi of five different genes (SEAP, DBI, PLK,Firefly Luciferase, and Renila Luciferase) by varying numbers ofrandomly selected siRNA and four rationally designed (SMART-selected)siRNA chosen using the algorithm described in Formula VIII. In addition,RNAi induced by a pool of the four SMART-selected siRNA is reported attwo different concentrations (100 and 400 nM). 10F is a comparisonbetween a pool of randomly selected EGFR siRNA (Pool 1) and a pool ofSMART selected EGFR siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4represent the individual members that made up each respective pool. Notethat numbers for random siRNAs represent the position of the 5′ end ofthe sense strand of the duplex. The Y-axis represents the % expressionof the control(s). The X-axis is the percent expression of the control.

FIG. 11 shows the Western blot results from cells treated with siRNAdirected against twelve different genes involved in theclathrin-dependent endocytosis pathway (CHC, DynII, CALM, CLCa, CLCb,Eps15, Eps15R, Rab5a, Rab5b, Rab5c, β2 subunit of AP-2 and EEA.1). siRNAwere selected using Formula VIII. “Pool” represents a mixture ofduplexes 1-4. Total concentration of each siRNA in the pool is 25 nM.Total concentration=4×25=100 nM.

FIG. 12 is a representation of the gene silencing capabilities ofrationally-selected siRNA directed against ten different genes (humanand mouse cyclophilin, C-myc, human lamin A/C, QB (ubiquinol-cytochromec reductase core protein I), MEK1 and MEK2, ATE1 (arginyl-tRNA proteintransferase), GAPDH, and Eg5). The Y-axis is the percent expression ofthe control. Numbers 1, 2, 3 and 4 represent individual rationallyselected siRNA. “Pool” represents a mixture of the four individualsiRNA.

FIG. 13 is the sequence of the top ten Bcl2 siRNAs as determined byFormula VIII. Sequences are listed 5′ to 3′.

FIG. 14 is the knockdown by the top ten Bcl2 siRNAs at 100 nMconcentrations. The Y-axis represents the amount of expression relativeto the non-specific (ns) and transfection mixture control.

FIG. 15 represents a functional walk where siRNA beginning on everyother base pair of a region of the luciferase gene are tested for theability to silence the luciferase gene. The Y-axis represents thepercent expression relative to a control. The X-axis represents theposition of each individual siRNA.

FIG. 16 is a histogram demonstrating the inhibition of target geneexpression by pools of 2 and 3 siRNAs duplexes taken from the walkdescribed in FIG. 15. The Y-axis represents the percent expressionrelative to control. The X-axis represents the position of the firstsiRNA in paired pools, or trios of siRNA. For instance, the first pairedpool contains siRNA 1 and 3. The second paired pool contains siRNA 3 and5. Pool 3 (of paired pools) contains siRNA 5 and 7, and so on.

FIG. 17 is a histogram demonstrating the inhibition of target geneexpression by pools of 4 and 5 siRNA duplexes. The Y-axis represents thepercent expression relative to a control. The X-axis represents theposition of the first siRNA in each pool.

FIG. 18 is a histogram demonstrating the inhibition of target geneexpression by siRNAs that are ten and twenty basepairs apart. The Y-axisrepresents the percent expression relative to a control. The X-axisrepresents the position of the first siRNA in each pool.

FIG. 19 shows that pools of siRNAs (dark gray bar) work as well (orbetter) than the best siRNA in the pool (light gray bar). The Y-axisrepresents the percent expression relative to a control. The-X axisrepresents the position of the first siRNA in each pool.

FIG. 20 shows that the combination of several semifunctional siRNAs(dark gray) result in a significant improvement of gene expressioninhibition over individual (semi-functional; light gray) siRNA. TheY-axis represents the percent expression relative to a control.

FIG. 21 shows both pools (Library, Lib) and individual siRNAs ininhibition of gene expression of Beta-Galactosidase, Renilla Luciferaseand SEAP (alkaline phosphatase). Numbers on the X-axis indicate theposition of the 5′-most nucleotide of the sense strand of the duplex.The Y-axis represents the percent expression of each gene relative to acontrol. Libraries contain 19 nucleotide long siRNAs (not includingoverhangs) that begin at the following nucleotides: SEAP: Lib 1: 206,766, 812,923, Lib 2: 1117, 1280, 1300, 1487, Lib 3: 206, 766, 812, 923,1117, 1280, 1300,1487, Lib 4: 206, 812, 1117, 1300, Lib 5: 766, 923,1280, 1487, Lib 6: 206, 1487; Bgal: Lib 1: 979, 1339, 2029, 2590, Lib 2:1087,1783,2399,3257, Lib 3: 979, 1783, 2590, 3257, Lib 4: 979, 1087,1339, 1783, 2029, 2399,2590,3257, Lib 5: 979, 1087, 1339, 1783, Lib 6:2029,2399,2590,3257; Renilla: Lib 1: 174,300,432,568, Lib 2: 592, 633,729,867, Lib 3: 174, 300, 432, 568, 592, 633,729,867, Lib 4: 174, 432,592, 729, Lib 5: 300,568,633,867, Lib 6: 592,568.

FIG. 22 shows the results of an EGFR and TfnR internalization assay whensingle gene knockdowns are performed. The Y-axis represents percentinternalization relative to control.

FIG. 23 shows the results of an EGFR and TfnR internalization assay whenmultiple genes are knocked down (e.g., Rab5a, b, c). The Y-axisrepresents the percent internalization relative to control.

FIG. 24 shows the simultaneous knockdown of four different genes. siRNAsdirected against G6PD, GAPDH, PLK, and UQCwere simultaneously introducedinto cells. Twenty-four hours later, cultures were harvested and assayedfor mRNA target levels for each of the four genes. A comparison is madebetween cells transfected with individual siRNAs vs. a pool of siRNAsdirected against all four genes.

FIG. 25 shows the functionality of ten siRNAs at 0.3 nM concentrations.

DETAILED DESCRIPTION DEFINITIONS

Unless stated otherwise, the following terms and phrases have themeanings provided below:

siRNA

The term “siRNA” refers to small inhibitory RNA duplexes that induce theRNA interference (RNAi) pathway. These molecules can vary in length(generally 18-30 basepairs) and contain varying degrees ofcomplementarity to their target mRNA in the antisense strand. Some, butnot all, siRNA have unpaired overhanging bases on the 5′ or 3′ end ofthe sense strand and/or the antisense strand. The term “siRNA” includesduplexes of two separate strands, as well as single strands that canform hairpin structures comprising a duplex region.

siRNA may be divided into five (5) groups (non-functional,semi-functional, functional, highly functional, and hyper-functional)based on the level or degree of silencing that they induce in culturedcell lines. As used herein, these definitions are based on a set ofconditions where the siRNA is transfected into said cell line at aconcentration of 100 nM and the level of silencing is tested at a timeof roughly 24 hours after transfection, and not exceeding 72 hours aftertransfection. In this context, “non-functional siRNA” are defined asthose siRNA that induce less than 50% (<50%) target silencing.“Semi-functional siRNA” induce 50-79% target silencing. “FunctionalsiRNA” are molecules that induce 80-95% gene silencing.“Highly-functional siRNA” are molecules that induce greater than 95%gene silencing. “Hyperfunctional siRNA” are a special class ofmolecules. For purposes of this document, hyperfunctional siRNA aredefined as those molecules that: (1) induce greater than 95% silencingof a specific target when they are transfected at subnanomolarconcentrations (i.e., less than one nanomolar); and/or (2) inducefunctional (or better) levels of silencing for greater than 96 hours.These relative functionalities (though not intended to be absolutes) maybe used to compare siRNAs to a particular target for applications suchas functional genomics, target identification and therapeutics.

miRNA

The term “miRNA” refers to microRNA.

Gene Silencing

The phrase “gene silencing” refers to a process by which the expressionof a specific gene product is lessened or attenuated. Gene silencing cantake place by a variety of pathways. Unless specified otherwise, as usedherin, gene silencing refers to decreases in gene product expressionthat results from RNA interference (RNAi), a defined, though partiallycharacterized pathway whereby small inhibitory RNA (siRNA) act inconcert with host proteins (e.g., the RNA induced silencing complex,RISC) to degrade messenger RNA (mRNA) in a sequence-dependent fashion.The level of gene silencing can be measured by a variety of means,including, but not limited to, measurement of transcript levels byNorthern Blot Analysis, B-DNA techniques, transcription-sensitivereporter constructs, expression profiling (e.g., DNA chips), and relatedtechnologies. Alternatively, the level of silencing can be measured byassessing the level of the protein encoded by a specific gene. This canbe accomplished by performing a number of studies including WesternAnalysis, measuring the levels of expression of a reporter protein thathas e.g., fluorescent properties (e.g., GFP) or enzymatic activity(e.g., alkaline phosphatases), or several other procedures.

Filters

The term “filter” refers to one or more procedures that are performed onsequences that are identified by the algorithm. In some instances,filtering includes in silico procedures where sequences identified bythe algorithm can be screened to identify duplexes carrying desirable orundesirable motifs. Sequences carrying such motifs can be selected for,or selected against, to obtain a final set with the preferredproperties. In other instances, filtering includes wet lab experiments.For instance, sequences identified by one or more versions of thealgorithm can be screened using any one of a number of procedures toidentify duplexes that have hyperfunctional traits (e.g., they exhibit ahigh degree of silencing at subnanomolar concentrations and/or exhibithigh degrees of silencing longevity).

Transfection

The term “transfection” refers to a process by which agents areintroduced into a cell. The list of agents that can be transfected islarge and includes, but is not limited to, siRNA, sense and/oranti-sense sequences, DNA encoding one or more genes and organized intoan expression plasmid, proteins, protein fragments, and more. There aremultiple methods for transfecting agents into a cell including, but notlimited to, electroporation, calcium phosphate-based transfections,DEAE-dextran-based transfections, lipid-based transfections, molecularconjugate-based transfections (e.g., polylysine-DNA conjugates),microinjection and others.

Target

The term “target” is used in a variety of different forms throughoutthis document and is defined by the context in which it is used. “TargetmRNA” refers to a messenger RNA to which a given siRNA can be directedagainst. “Target sequence” and “target site” refer to a sequence withinthe mRNA to which the sense strand of an siRNA shows varying degrees ofhomology and the antisense strand exhibits varying degrees ofcomplementarity. The phrase “siRNA target” can refer to the gene, mRNA,or protein against which an siRNA is directed. Similarly, “targetsilencing” can refer to the state of a gene, or the corresponding mRNAor protein.

Off-Target Silencing and Off-Target Interference

The phrases “off-target silencing” and “off-target interference” aredefined as degradation of mRNA other than the intended target mRNA dueto overlapping and/or partial homology with secondary mRNA messages.

SMARTscore™

The term “SMARTscore™” refers to a number determined by applying any ofthe Formulas I-Formula X to a given siRNA sequence. The phrases“SMART-selected” or “rationally selected” or “rational selection” referto siRNA that have been selected on the basis of their SMARTscores™.

Complementary

The term “complementary” refers to the ability of polynucleotides toform base pairs with one another. Base pairs are typically formed byhydrogen bonds between nucleotide units in antiparallel polynucleotidestrands. Complementary polynucleotide strands can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G), or in any othermanner that allows for the formation of duplexes. As persons skilled inthe art are aware, when using RNA as opposed to DNA, uracil rather thanthymine is the base that is considered to be complementary to adenosine.However, when a U is denoted in the context of the present invention,the ability to substitute a T is implied, unless otherwise stated.

Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can hydrogenbond with a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can hydrogen bond with each other.For example, for two 20-mers, if only two base pairs on each strand canhydrogen bond with each other, the polynucleotide strands exhibit 10%complementarity. In the same example, if 18 base pairs on each strandcan hydrogen bond with each other, the polynucleotide strands exhibit90% complementarity. “Substantial complementarity” refers topolynucleotide strands exhibiting 79% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. (“Substantial similarity”refers to polynucleotide strands exhibiting 79% or greater similarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as not to be similar.) Thus, for example, twopolynucleotides of 29 nucleotide units each, wherein each comprises adi-dT at the 3′ terminus such that the duplex region spans 27 bases, andwherein 26 of the 27 bases of the duplex region on each strand arecomplementary, are substantially complementary since they are 96.3%complementary when excluding the di-dT overhangs.

Deoxynucleotide

The term “deoxynucleotide” refers to a nucleotide or polynucleotidelacking a hydroxyl group (OH group) at the 2′ and/or 3′ position of asugar moiety. Instead, it has a hydrogen bonded to the 2′ and/or 3′carbon. Within an RNA molecule that comprises one or moredeoxynucleotides, “deoxynucleotide” refers to the lack of an OH group atthe 2′ position of the sugar moiety, having instead a hydrogen bondeddirectly to the 2′ carbon.

Deoxyribonucleotide

The terms “deoxyribonucleotide” and “DNA” refer to a nucleotide orpolynucleotide comprising at least one sugar moiety that has an H,rather than an OH, at its 2′ and/or 3′ position.

Substantially Similar

The phrase “substantially similar” refers to a similarity of at least90% with respect to the identity of the bases of the sequence.

Duplex Region

The phrase “duplex region” refers to the region in two complementary orsubstantially complementary polynucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for a stabilized duplex between polynucleotide strands thatare complementary or substantially complementary. For example, apolynucleotide strand having 21 nucleotide units can base pair withanother polynucleotide of 21 nucleotide units, yet only 19 bases on eachstrand are complementary or substantially complementary, such that the“duplex region” has 19 base pairs. The remaining bases may, for example,exist as 5′ and 3′ overhangs. Further, within the duplex region, 100%complementarity is not required; substantial complementarity isallowable within a duplex region. Substantial complementarity refers to79% or greater complementarity. For example, a mismatch in a duplexregion consisting of 19 base pairs results in 94.7% complementarity,rendering the duplex region substantially complementary.

Nucleotide

The term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide or modified form thereof, as well as an analogthereof. Nucleotides include species that comprise purines, e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs, aswell as pyrimidines, e.g., cytosine, uracil, thymine, and theirderivatives and analogs.

Nucleotide analogs include nucleotides having modifications in thechemical structure of the base, sugar and/or phosphate, including, butnot limited to, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at cytosine exocyclic amines, andsubstitution of 5-bromo-uracil; and 2′-position sugar modifications,including but not limited to, sugar-modified ribonucleotides in whichthe 2′—OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH₂,NHR, NR₂, or CN, wherein R is an alkyl moiety. Nucleotide analogs arealso meant to include nucleotides with bases such as inosine, queuosine,xanthine, sugars such as 2′-methyl ribose, non-natural phosphodiesterlinkages such as methylphosphonates, phosphorothioates and peptides.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of types of modifications that cancomprise nucleotides that are modified with respect to the base moietiesinclude but are not limited to, alkylated, halogenated, thiolated,aminated, amidated, or acetylated bases, individually or in combination.More specific examples include, for example, 5-propynyluridine,5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose,and other sugars, heterocycles, or carbocycles.

The term nucleotide is also meant to include what are known in the artas universal bases. By way of example, universal bases include but arenot limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term“nucleotide” is also meant to include the N3′ to P5′ phosphoramidate,resulting from the substitution of a ribosyl 3′ oxygen with an aminegroup.

Further, the term nucleotide also includes those species that have adetectable label, such as for example a radioactive or fluorescentmoiety, or mass label attached to the nucleotide.

Polynucleotide

The term “polynucleotide” refers to polymers of nucleotides, andincludes but is not limited to DNA, RNA, DNA/RNA hybrids includingpolynucleotide chains of regularly and/or irregularly alternatingdeoxyribosyl moieties and ribosyl moieties (i.e., wherein alternatenucleotide units have an —OH, then and —H, then an —OH, then an —H, andso on at the 2′ position of a sugar moiety), and modifications of thesekinds of polynucleotides, wherein the attachment of various entities ormoieties to the nucleotide units at any position are included.

Polyribonucleotide

The term “polyribonucleotide” refers to a polynucleotide comprising twoor more modified or unmodified ribonucleotides and/or their analogs. Theterm “polyribonucleotide” is used interchangeably with the term“oligoribonucleotide.”

Ribonucleotide and Ribonucleic Acid

The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), referto a modified or unmodified nucleotide or polynucleotide comprising atleast one ribonucleotide unit. A ribonucleotide unit comprises anhydroxyl group attached to the 2′ position of a ribosyl moiety that hasa nitrogenous base attached in N-glycosidic linkage at the 1′ positionof a ribosyl moiety, and a moiety that either allows for linkage toanother nucleotide or precludes linkage.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to improving the efficiency of genesilencing by siRNA. Through the inclusion of multiple siRNA sequencesthat are targeted to a particular gene and/or selecting an siRNAsequence based on certain defined criteria, improved efficiency may beachieved.

The present invention will now be described in connection with preferredembodiments. These embodiments are presented in order to aid in anunderstanding of the present invention and are not intended, and shouldnot be construed, to limit the invention in any way. All alternatives,modifications and equivalents that may become apparent to those ofordinary skill upon reading this disclosure are included within thespirit and scope of the present invention.

Furthermore, this disclosure is not a primer on RNA interference. Basicconcepts known to persons skilled in the art have not been set forth indetail.

The present invention is directed to increasing the efficiency of RNAi,particularly in mammalian systems. Accordingly, the present inventionprovides kits, siRNAs and methods for increasing siRNA efficacy.

According to a first embodiment, the present invention provides a kitfor gene silencing, wherein said kit is comprised of a pool of at leasttwo siRNA duplexes, each of which is comprised of a sequence that iscomplementary to a portion of the sequence of one or more targetmessenger RNA, and each of which is selected using non-target specificcriteria. Each of the at least two siRNA duplexes of the kitcomplementary to a portion of the sequence of one or more target mRNAsis preferably selected using Formula X.

According to a second embodiment, the present invention provides amethod for selecting an siRNA, said method comprising applying selectioncriteria to a set of potential siRNA that comprise 18-30 base pairs,wherein said selection criteria are non-target specific criteria, andsaid set comprises at least two siRNAs and each of said at least twosiRNAs contains a sequence that is at least substantially complementaryto a target gene; and determining the relative functionality of the atleast two siRNAs.

In one embodiment, the present invention also provides a method whereinsaid selection criteria are embodied in a formula comprising:(−14)*G₁₃−13*A₁−12*U₇−11*U₂−10*A₁₁−10*U₄−10*C₃−10*C₅−10*C₆−9*A₁₀−9*U₉−9*C₁₈−8*G₁₀−7*U₁−7*U₁₆−7*C₁₇−7*C₁₉+7*U₁₇+8*A₂+8*A₄+8*A₅+8*C₄+9*G₈+10*A₇+10*U₁₈+11*A₁₉+11*C₉+15*G₁+18*A₃+19*U₁₀−Tm−3*(GC_(total))−6*(GC₁₅₋₁₉)−30*X; or   Formula VIII(−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(numberof A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula Xwherein position numbering begins at the 5′-most position of a sensestrand, and

-   -   A₁=1 if A is the base at position 1 of the sense strand,        otherwise its value is 0;    -   A₂=1 if A is the base at position 2 of the sense strand,        otherwise its value is 0;    -   A₃=1 if A is the base at position 3 of the sense strand,        otherwise its value is 0;    -   A₄=1 if A is the base at position 4 of the sense strand,        otherwise its value is 0;    -   A₅=1 if A is the base at position 5 of the sense strand,        otherwise its value is 0;    -   A₆=1 if A is the base at position 6 of the sense strand,        otherwise its value is 0;    -   A₇=1 if A is the base at position 7 of the sense strand,        otherwise its value is 0;    -   A₁₀=1 if A is the base at position 10 of the sense strand,        otherwise its value is 0;    -   A₁₁=1 if A is the base at position 11 of the sense strand,        otherwise its value is 0;    -   A₁₃=1 if A is the base at position 13 of the sense strand,        otherwise its value is 0;    -   A₁₉=1 if A is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   C₃=1 if C is the base at position 3 of the sense strand,        otherwise its value is 0;    -   C₄=1 if C is the base at position 4 of the sense strand,        otherwise its value is 0;    -   C₅=1 if C is the base at position 5 of the sense strand,        otherwise its value is 0;    -   C₆=1 if C is the base at position 6 of the sense strand,        otherwise its value is 0;    -   C₇=1 if C is the base at position 7 of the sense strand,        otherwise its value is 0;    -   C₉=1 if C is the base at position 9 of the sense strand,        otherwise its value is 0;    -   C₁₇=1 if C is the base at position 17 of the sense strand,        otherwise its value is 0;    -   C₁₈=1 if C is the base at position 18 of the sense strand,        otherwise its value is 0;    -   C₁₉=1 if C is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   G₁=1 if G is the base at position 1 on the sense strand,        otherwise its value is 0;    -   G₂=1 if G is the base at position 2 of the sense strand,        otherwise its value is 0;    -   G₈=1 if G is the base at position 8 on the sense strand,        otherwise its value is 0;    -   G₁₀=1 if G is the base at position 10 on the sense strand,        otherwise its value is 0;    -   G₁₃=1 if G is the base at position 13 on the sense strand,        otherwise its value is 0;    -   G₁₉=1 if G is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   U₁=1 if U is the base at position 1 on the sense strand,        otherwise its value is 0;    -   U₂=1 if U is the base at position 2 on the sense strand,        otherwise its value is 0;    -   U₃=1 if U is the base at position 3 on the sense strand,        otherwise its value is 0;    -   U₄=1 if U is the base at position 4 on the sense strand,        otherwise its value is 0;    -   U₇=1 if U is the base at position 7 on the sense strand,        otherwise its value is 0;    -   U₉=1 if U is the base at position 9 on the sense strand,        otherwise its value is 0;    -   U ₁₀=1 if U is the base at position 10 on the sense strand,        otherwise its value is 0;    -   U₁₅=1 if U is the base at position 15 on the sense strand,        otherwise its value is 0;    -   U₁₆=1 if U is the base at position 16 on the sense strand,        otherwise its value is 0;    -   U₁₇=1 if U is the base at position 17 on the sense strand,        otherwise its value is 0;    -   U₁₈=1 if U is the base at position 18 on the sense strand,        otherwise its value is 0.    -   GC₁₅₋₁₉=the number of G and C bases within positions 15-19 of        the sense strand, or within positions 15-18 if the sense strand        is only 18 base pairs in length;    -   GC_(total)=the number of G and C bases in the sense strand;    -   Tm=100 if the siRNA oligo has the internal repeat longer then 4        base pairs, otherwise its value is 0; and    -   X=the number of times that the same nucleotide repeats four or        more times in a row.

Any of the methods of selecting siRNA in accordance with the inventioncan further comprise comparing the internal stability profiles of thesiRNAs to be selected, and selecting those siRNAs with the mostfavorable internal stability profiles. Any of the methods of selectingsiRNA can further comprise selecting either for or against sequencesthat contain motifs that induce cellular stress. Such motifs include,for example, toxicity motifs. Any of the methods of selecting siRNA canfurther comprise either selecting for or selecting against sequencesthat comprise stability motifs.

In another embodiment, the present invention provides a method of genesilencing, comprising introducing into a cell at least one siRNAselected according to any of the methods of the present invention. ThesiRNA can be introduced by allowing passive uptake of siRNA, or throughthe use of a vector.

According to a third embodiment, the invention provides a method fordeveloping an algorithm for selecting siRNA, said method comprising: (a)selecting a set of siRNA; (b) measuring gene silencing ability of eachsiRNA from said set; (c) determining relative functionality of eachsiRNA; (d) determining improved functionality by the presence or absenceof at least one variable selected from the group consisting of thepresence or absence of a particular nucleotide at a particular position,the total number of As and Us in positions 15-19, the number of timesthat the same nucleotide repeats within a given sequence, and the totalnumber of Gs and Cs; and (e) developing an algorithm using theinformation of step (d).

In another embodiment, the invention provides a method for selecting ansiRNA with improved functionality, comprising using the above-mentionedalgorithm to identify an siRNA of improved functionality.

According to a fourth embodiment, the present invention provides a kit,wherein said kit is comprised of at least two siRNAs, wherein said atleast two siRNAs comprise a first optimized siRNA and a second optimizedsiRNA, wherein said first optimized siRNA and said second optimizedsiRNA are optimized according a formula comprising Formula X.

According to a fifth embodiment, the present invention provides a methodfor identifying a hyperfunctional siRNA, comprising applying selectioncriteria to a set of potential siRNA that comprise 18-30 base pairs,wherein said selection criteria are non-target specific criteria, andsaid set comprises at least two siRNAs and each of said at least twosiRNAs contains a sequence that is at least substantially complementaryto a target gene; determining the relative functionality of the at leasttwo siRNAs and assigning each of the at least two siRNAs a functionalityscore; and selecting siRNAs from the at least two siRNAs that have afunctionality score that reflects greater than 80 percent silencing at aconcentration in the picomolar range, wherein said greater than 80percent silencing endures for greater than 120 hours.

In other embodiments, the invention provides kits and/or methods whereinthe siRNA are comprised of two separate polynucleotide strands; whereinthe siRNA are comprised of a single contiguous molecule such as, forexample, a unimolecular siRNA (comprising, for example, either anucleotide or non-nucleotide loop); wherein the siRNA are expressed fromone or more vectors; and wherein two or more genes are silenced by asingle administration of siRNA.

According to a sixth embodiment, the present invention provides ahyperfunctional siRNA that is capable of silencing Bc12.

According to a seventh embodiment, the present invention provides amethod for developing an siRNA algorithm for selecting functional andhyperfunctional siRNAs for a given sequence. The method comprises:

-   -   (a) selecting a set of siRNAs;    -   (b) measuring the gene silencing ability of each siRNA from said        set;    -   (c) determining the relative functionality of each siRNA;    -   (d) determining the amount of improved functionality by the        presence or absence of at least one variable selected from the        group consisting of the total GC content, melting temperature of        the siRNA, GC content at positions 15-19, the presence or        absence of a particular nucleotide at a particular position,        relative thermodynamic stability at particular positions in a        duplex, and the number of times that the same nucleotide repeats        within a given sequence; and    -   (e) developing an algorithm using the information of step (d).        According to this embodiment, preferably the set of siRNAs        comprises at least 90 siRNAs from at least one gene, more        preferably at least 180 siRNAs from at least two different        genes, and most preferably at least 270 and 360 siRNAs from at        least three and four different genes, respectively.        Additionally, in step (d) the determination is made with        preferably at least two, more preferably at least three, even        more preferably at least four, and most preferably all of the        variables. The resulting algorithm is not target sequence        specific.

In another embodiment, the present invention provides rationallydesigned siRNAs identified using the formulas above.

In yet another embodiment, the present invention is directed tohyperfunctional siRNA.

The ability to use the above algorithms, which are not sequence orspecies specific, allows for the cost-effective selection of optimizedsiRNAs for specific target sequences. Accordingly, there will be bothgreater efficiency and reliability in the use of siRNA technologies.

The methods disclosed herein can be used in conjunction with comparinginternal stability profiles of selected siRNAs, and designing an siRNAwith a desireable internal stability profile; and/or in conjunction witha selection either for or against sequences that contain motifs thatinduce cellular stress, for example, cellular toxicity.

Any of the methods disclosed herein can be used to silence one or moregenes by introducing an siRNA selected, or designed, in accordance withany of the methods disclosed herein. The siRNA(s) can be introduced intothe cell by any method known in the art, including passive uptake orthrough the use of one or more vectors.

Any of the methods and kits disclosed herein can employ eitherunimolecular siRNAs, siRNAs comprised of two separate polynucleotidestrands, or combinations thereof. Any of the methods disclosed hereincan be used in gene silencing, where two or more genes are silenced by asingle administration of siRNA(s). The siRNA(s) can be directed againsttwo or more target genes, and administered in a single dose or singletransfection, as the case may be.

Optimizing siRNA

According to one embodiment, the present invention provides a method forimproving the effectiveness of gene silencing for use to silence aparticular gene through the selection of an optimal siRNA. An siRNAselected according to this method may be used individually, or inconjunction with the first embodiment, i.e., with one or more othersiRNAs, each of which may or may not be selected by this criteria inorder to maximize their efficiency.

The degree to which it is possible to select an siRNA for a given mRNAthat maximizes these criteria will depend on the sequence of the mRNAitself. However, the selection criteria will be independent of thetarget sequence. According to this method, an siRNA is selected for agiven gene by using a rational design. That said, rational design can bedescribed in a variety of ways. Rational design is, in simplest terms,the application of a proven set of criteria that enhance the probabilityof identifying a functional or hyperfunctional siRNA. In one method,rationally designed siRNA can be identified by maximizing one or more ofthe following criteria:

-   -   1. A low GC content, preferably between about 30-52%.    -   2. At least 2, preferably at least 3 A or U bases at positions        15-19 of the siRNA on the sense strand.    -   3. An A base at position 19 of the sense strand.    -   4. An A base at position 3 of the sense strand.    -   5. A U base at position 10 of the sense strand.    -   6. An A base at position 14 of the sense strand.    -   7. A base other than C at position 19 of the sense strand.    -   8. A base other than G at position 13 of the sense strand.    -   9. A Tm, which refers to the character of the internal repeat        that results in inter- or intramolecular structures for one        strand of the duplex, that is preferably not stable at greater        than 50° C., more preferably not stable at greater than 37° C.,        even more preferably not stable at greater than 30° C. and most        preferably not stable at greater than 20° C.    -   10. A base other than U at position 5 of the sense strand.    -   11. A base other than A at position 11 of the sense strand.    -   12. A base other than an A at position 1 of the sense strand.    -   13. A base other than an A at position 2 of the sense strand.    -   14. An A base at position 4 of the sense strand.    -   15. An A base at position 5 of the sense strand.    -   16. An A base at position 6 of the sense strand.    -   17. An A base at position 7 of the sense strand.    -   18. An A base at position 8 of the sense strand.    -   19. A base other than an A at position 9 of the sense strand.    -   20. A base other than an A at position 10 of the sense strand.    -   21. A base other than an A at position 11 of the sense strand.    -   22. A base other than an A at position 12 of the sense strand.    -   23. An A base at position 13 of the sense strand.    -   24. A base other than an A at position 14 of the sense strand.    -   25. An A base at position 15 of the sense strand    -   26. An A base at position 16 of the sense strand.    -   27. An A base at position 17 of the sense strand.    -   28. An A base at position 18 of the sense strand.    -   29. A base other than a U at position 1 of the sense strand.    -   30. A base other than a U at position 2 of the sense strand.    -   31. A U base at position 3 of the sense strand.    -   32. A base other than a U at position 4 of the sense strand.    -   33. A base other than a U at position 5 of the sense strand.    -   34. A U base at position 6 of the sense strand.    -   35. A base other than a U at position 7 of the sense strand.    -   36. A base other than a U at position 8 of the sense strand.    -   37. A base other than a U at position 9 of the sense strand.    -   38. A base other than a U at position 11 of the sense strand.    -   39. A U base at position 13 of the sense strand.    -   40. A base other than a U at position 14 of the sense strand.    -   41. A base other than a U at position 15 of the sense strand.    -   42. A base other than a U at position 16 of the sense strand.    -   43. A U base at position 17 of the sense strand.    -   44. A U base at position 18 of the sense strand.    -   45. A U base at position 19 of the sense strand.    -   46. A C base at position 1 of the sense strand.    -   47. A C base at position 2 of the sense strand.    -   48. A base other than a C at position 3 of the sense strand.    -   49. A C base at position 4 of the sense strand.    -   50. A base other than a C at position 5 of the sense strand.    -   51. A base other than a C at position 6 of the sense strand.    -   52. A base other than a C at position 7 of the sense strand.    -   53. A base other than a C at position 8 of the sense strand.    -   54. A C base at position 9 of the sense strand.    -   55. A C base at position 10 of the sense strand.    -   56. A C base at position 11 of the sense strand.    -   57. A base other than a C at position 12 of the sense strand.    -   58. A base other than a C at position 13 of the sense strand.    -   59. A base other than a C at position 14 of the sense strand.    -   60. A base other than a C at position 15 of the sense strand.    -   61. A base other than a C at position 16 of the sense strand.    -   62. A base other than a C at position 17 of the sense strand.    -   63. A base other than a C at position 18 of the sense strand.    -   64. A G base at position 1 of the sense strand.    -   65. A G base at position 2 of the sense strand.    -   66. A G base at position 3 of the sense strand.    -   67. A base other than a G at position 4 of the sense strand.    -   68. A base other than a G at position 5 of the sense strand.    -   69. A G base at position 6 of the sense strand.    -   70. A G base at position 7 of the sense strand.    -   71. A G base at position 8 of the sense strand.    -   72. A G base at position 9 of the sense strand.    -   73. A base other than a G at position 10 of the sense strand.    -   74. A G base at position 11 of the sense strand.    -   75. A G base at position 12 of the sense strand.    -   76. A G base at position 14 of the sense strand.    -   77. A G base at position 15 of the sense strand.    -   78. A G base at position 16 of the sense strand.    -   79. A base other than a G at position 17 of the sense strand.    -   80. A base other than a G at position 18 of the sense strand.    -   81. A base other than a G at position 19 of the sense strand.

The importance of various criteria can vary greatly. For instance, a Cbase at position 10 of the sense strand makes a minor contribution toduplex functionality. In contrast, the absence of a C at position 3 ofthe sense strand is very important. Accordingly, preferably an siRNAwill satisfy as many of the aforementioned criteria as possible.

With respect to the criteria, GC content, as well as a high number of AUin positions 15-19 of the sense strand, may be important for easement ofthe unwinding of double stranded siRNA duplex. Duplex unwinding has beenshown to be crucial for siRNA functionality in vivo.

With respect to criterion 9, the internal structure is measured in termsof the melting temperature of the single strand of siRNA, which is thetemperature at which 50% of the molecules will become denatured. Withrespect to criteria 2-8 and 10-11, the positions refer to sequencepositions on the sense strand, which is the strand that is identical tothe mRNA.

In one preferred embodiment, at least criteria 1 and 8 are satisfied. Inanother preferred embodiment, at least criteria 7 and 8 are satisfied.In still another preferred embodiment, at least criteria 1, 8 and 9 aresatisfied.

It should be noted that all of the aforementioned criteria regardingsequence position specifics are with respect to the 5′ end of the sensestrand. Reference is made to the sense strand, because most databasescontain information that describes the information of the mRNA. Becauseaccording to the present invention a chain can be from 18 to 30 bases inlength, and the aforementioned criteria assumes a chain 19 base pairs inlength, it is important to keep the aforementioned criteria applicableto the correct bases.

When there are only 18 bases, the base pair that is not present is thebase pair that is located at the 3′ of the sense strand. When there aretwenty to thirty bases present, then additional bases are added at the5′ end of the sense chain and occupy positions ⁻1 to ⁻11. Accordingly,with respect to SEQ. ID NO. 0001 NNANANNNNUCNAANNNNA and SEQ. ID NO.0028 GUCNNANANNNNUCNAANNNNA, both would have A at position 3, A atposition 5, U at position 10, C at position 11, A and position 13, A andposition 14 and A at position 19. However, SEQ. ID NO. 0028 would alsohave C at position −1, U at position −2 and G at position −3.

For a 19 base pair siRNA, an optimal sequence of one of the strands maybe represented below, where N is any base, A, C, G, or U:NNANANNNNUCNAANNNNA. SEQ. ID NO. 0001 NNANANNNNUGNAANNNNA. SEQ. ID NO.0001 NNANANNNNUUNAANNNNA. SEQ. ID NO. 0002 NNANANNNNUCNCANNNNA. SEQ. IDNO. 0003 NNANANNNNUGNCANNNNA. SEQ. ID NO. 0004 NNANANNNNUUNCANNNNA. SEQ.ID NO. 0005 NNANANNNNUCNUANNNNA. SEQ. ID NO. 0006 NNANANNNNUGNUANNNNA.SEQ. ID NO. 0007 NNANANNNNUUNUANNNNA. SEQ. ID NO. 0008NNANCNNNNUCNAANNNNA. SEQ. ID NO. 0010 NNANCNNNNUGNAANNNNA. SEQ. ID NO.0011 NNANCNNNNUUNAANNNNA. SEQ. ID NO. 0012 NNANCNNNNUCNCANNNNA. SEQ. IDNO. 0013 NNANCNNNNUGNCANNNNA. SEQ. ID NO. 0014 NNANCNNNNUUNCANNNNA. SEQ.ID NO. 0015 NANCNNNNUCNUANNNNA. SEQ. ID NO. 0016 NNANCNNNNUGNUANNNNA.SEQ. ID NO. 0017 NNANCNNNNUUNUANNNNA. SEQ. ID NO. 0018NNANGNNNNUCNAANNNNA. SEQ. ID NO. 0019 NNANGNNNNUGNAANNNNA. SEQ. ID NO.0020 NNANGNNNNUUNAANNNNA. SEQ. ID NO. 0021 NNANGNNNNUCNCANNNNA. SEQ. IDNO. 0022 NNANGNNNNUGNCANNNNA. SEQ. ID NO. 0023 NNANGNNNNUUNCANNNNA. SEQ.ID NO. 0024 NNANGNNNNUCNUANNNNA. SEQ. ID NO. 0025 NNANGNNNNUGNUANNNNA.SEQ. ID NO. 0026 NNANGNNNNNUNUANNNNA. SEQ. ID NO. 0027

In one embodiment, the sequence used as an siRNA is selected by choosingthe siRNA that score highest according to one of the following sevenalgorithms that are represented by Formulas I-VII:Relative functionality of siRNA=−(GC/3)+(AU ₁₅₋₁₉)−Tm_(20°C.))*3−(G₁₃)*3)−(C ₁₉)   Formula IRelative functionality of siRNA=−(GC/3)−(AU ₁₅₋₁₉)*3−(G ₁₃)*3−(C ₁₉)+(A₁₉)*2+(A ₃)   Formula IIRelative functionality of siRNA=−(GC/3)+(AU ₁₅₋₁₉)−(Tm_(20°C.))*3  Formula IIIRelative functionality of siRNA=GC/2+(AU ₁₅₋₁₉)/2−(Tm_(20°C.))*2−(G₁₃)*3−(C₁₉)+(A ₁₉)*2+(A ₃)+(U ₁₀)+(A ₁₄)−(U ₅)−(A ₁₁)   Formula IVRelative functionality of siRNA=−(G ₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)+(U₁₀)+(A ₁₄)−(U ₅)−(A ₁₁)   Formula VRelative functionality of siRNA=−(G ₁₃)*3−(C ₁₉)+(A ₁₉)*2+(A ₃)  Formula VIRelative functionality of siRNA=−(GC/2)+(AU ₁₅₋₁₉)/2−(Tm_(20°C.))*1−(G₁₃)*3−(C ₁₉)+(A ₁₉)*3+(A ₃)*3+(U ₁₀)/2+(A ₁₄)/2−(U ₅)/2−(A ₁₁)/2  Formula VIIIn Formulas I-VII:

-   -   wherein A₁₉=1 if A is the base at position 19 on the sense        strand, otherwise its value is 0,        -   AU₁₅₋₁₉=0-5 depending on the number of A or U bases on the            sense strand at positions 15-19;        -   G₁₃=1 if G is the base at position 13 on the sense strand,            otherwise its value is 0;        -   C₁₉=1 if C is the base at position 19 of the sense strand,            otherwise its value is 0;        -   GC=the number of G and C bases in the entire sense strand;        -   Tm_(20° C.)=1 if the Tm is greater than 20° C.;        -   A₃=1 if A is the base at position 3 on the sense strand,            otherwise its value is 0;        -   U₁₀=1 if U is the base at position 10 on the sense strand,            otherwise its value is 0;        -   A₁₄=1 if A is the base at position 14 on the sense strand,            otherwise its value is 0;        -   U₅=1 if U is the base at position 5 on the sense strand,            otherwise its value is 0; and        -   A₁₁=1 if A is the base at position 11 of the sense strand,            otherwise its value is 0.

Formulas I-VII provide relative information regarding functionality.When the values for two sequences are compared for a given formula, therelative functionality is ascertained; a higher positive numberindicates a greater functionality. For example, in many applications avalue of 5 or greater is beneficial.

Additionally, in many applications, more than one of these formulaswould provide useful information as to the relative functionality ofpotential siRNA sequences. However, it is beneficial to have more thanone type of formula, because not every formula will be able to help todifferentiate among potential siRNA sequences. For example, inparticularly high GC mRNAs, formulas that take that parameter intoaccount would not be useful and application of formulas that lack GCelements (e.g., formulas V and VI) might provide greater insights intoduplex functionality. Similarly, formula II might by used in situationswhere hairpin structures are not observed in duplexes, and formula IVmight be applicable for sequences that have higher AU content. Thus, onemay consider a particular sequence in light of more than one or even allof these algorithms to obtain the best differentiation among sequences.In some instances, application of a given algorithim may identify anunususally large number of potential siRNA sequences, and in thosecases, it may be appropriate to re-analyze that sequence with a secondalgorithm that is, for instance, more stringent. Alternatively, it isconceivable that analysis of a sequence with a given formula yields noacceptable siRNA sequences (i.e., low SMARTscores™). In this instance,it may be appropriate to re-analyze that sequences with a secondalgorithm that is, for instance, less stringent. In still otherinstances, analysis of a single sequence with two separate formulas maygive rise to conflicting results (i.e., one formula generates a set ofsiRNA with high SMARTscores™ while the other formula identifies a set ofsiRNA with low SMARTscores™). In these instances, it may be necessary todetermine which weighted factor(s) (e.g., GC content) are contributingto the discrepancy and assessing the sequence to decide whether thesefactors should or should not be included. Alternatively, the sequencecould be analyzed by a third, fourth, or fifth algorithm to identify aset of rationally designed siRNA.

The above-referenced criteria are particularly advantageous when used incombination with pooling techniques as depicted in Table I: TABLE IFunctional Probability Oligos Pools Criteria >95% >80% <70% >95% >80%<70% Current 33.0 50.0 23.0 79.5 97.3 0.3 New 50.0 88.5 8.0 93.8 99.980.005 (GC) 28.0 58.9 36.0 72.8 97.1 1.6The term “current” used in Table I refers to Tuschl's conventional siRNAparameters (Elbashir, S. M. et al. (2002) “Analysis of gene function insomatic mammalian cells using small interfering RNAs” Methods 26:199-213). “New” refers to the design parameters described in FormulasI-VII. “GC” refers to criteria that select siRNA solely on the basis ofGC content.

As Table I indicates, when more functional siRNA duplexes are chosen,siRNAs that produce <70% silencing drops from 23% to 8% and the numberof siRNA duplexes that produce >80% silencing rises from 50% to 88.5%.Further, of the siRNA duplexes with >80% silencing, a larger portion ofthese siRNAs actually silence >95% of the target expression (the newcriteria increases the portion from 33% to 50%). Using this new criteriain pooled siRNAs, shows that, with pooling, the amount of silencing >95%increases from 79.5% to 93.8% and essentially eliminates any siRNA poolfrom silencing less than 70%.

Table II similarly shows the particularly beneficial results of poolingin combination with the aforementioned criteria. However, Table II,which takes into account each of the aforementioned variables,demonstrates even a greater degree of improvement in functionality.TABLE II Functional Probability Oligos Pools Func- Non- Func- Non-tional Average functional tional Average functional Random 20 40 50 6797 3 Criteria 1 52 99 0.1 97 93 0.0040 Criteria 4 89 99 0.1 99 99 0.0000The terms “functional,” “Average,” and “Non-functional” used in TableII, refer to siRNA that exhibit >80%, >50%, and <50% functionality,respectively. Criteria 1 and 4 refer to specific criteria describedabove.

The above-described algorithms may be used with or without a computerprogram that allows for the inputting of the sequence of the mRNA andautomatically outputs the optimal siRNA. The computer program may, forexample, be accessible from a local terminal or personal computer, overan internal network or over the Internet.

In addition to the formulas above, more detailed algorithms may be usedfor selecting siRNA. Preferably, at least one RNA duplex of 18-30 basepairs is selected such that it is optimized according a formula selectedfrom:(−14)*G₁₃−13*A₁−12*U₇−11*U₂−10*A₁₁−10*U₄−10*C₃−10*C₅−10*C₆−9*A₁₀−9*U₉−9*C₁₈−8*G₁₀−7*U₁−7*U₁₆−7*C₁₇−7*C₁₉+7*U₁₇+8*A₂+8*A₄+8*A₅+8*C₄+9*G₈+10*A₇+10*U₁₈+11*A₁₉+11*C₉+15*G₁+18*A₃+19*U₁₀−Tm−3*(GC_(total))−6*(GC₁₅₋₁₉)−30*X; and   Formula VIII(14.1)*A₃+(14.9)*A₆+(17.6)*A₁₃+(24.7)*A₁₉+(14.2)*U₁₀+((10.5)*C₉+(23.9)*G₁+(16.3)*G₂+(−12.3)*A₁₁+(−19.3)*U₁+(−12.1)*U₂+(−11)*U₃+(−15.2)*U₁₅+(−11.3)*U₁₆+(−11.8)*C₃+(−17.4)*C₆+(−10.5)*C₇+(−13.7)*G₁₃+(−25.9)*G₁₉−Tm−3*(GC_(total))−6*(GC₁₅₋₁₉)−30*X;and   Formula IX(−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(numberof A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula Xwherein

-   -   A₁=1 if A is the base at position 1 of the sense strand,        otherwise its value is 0;    -   A₂=1 if A is the base at position 2 of the sense strand,        otherwise its value is 0;    -   A₃=1 if A is the base at position 3 of the sense strand,        otherwise its value is 0;    -   A₄=1 if A is the base at position 4 of the sense strand,        otherwise its value is 0;    -   A₅=1 if A is the base at position 5 of the sense strand,        otherwise its value is 0;    -   A₆=1 if A is the base at position 6 of the sense strand,        otherwise its value is 0;    -   A₇=1 if A is the base at position 7 of the sense strand,        otherwise its value is 0;    -   A₁₀=1 if A is the base at position 10 of the sense strand,        otherwise its value is 0;    -   A₁₁=1 if A is the base at position 11 of the sense strand,        otherwise its value is 0;    -   A₁₃=1 if A is the base at position 13 of the sense strand,        otherwise its value is 0;    -   A₁₉=1 if A is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   C₃=1 if C is the base at position 3 of the sense strand,        otherwise its value is 0;    -   C₄=1 if C is the base at position 4 of the sense strand,        otherwise its value is 0;    -   C₅=1 if C is the base at position 5 of the sense strand,        otherwise its value is 0;    -   C₆=1 if C is the base at position 6 of the sense strand,        otherwise its value is 0;    -   C₇=1 if C is the base at position 7 of the sense strand,        otherwise its value is 0;    -   C₉=1 if C is the base at position 9 of the sense strand,        otherwise its value is 0;    -   C₁₇=1 if C is the base at position 17 of the sense strand,        otherwise its value is 0;    -   C₁₈=1 if C is the base at position 18 of the sense strand,        otherwise its value is 0;    -   C₁₉=1 if C is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   G₁=1 if G is the base at position 1 on the sense strand,        otherwise its value is 0;    -   G₂=1 if G is the base at position 2 of the sense strand,        otherwise its value is 0;    -   G₈=1 if G is the base at position 8 on the sense strand,        otherwise its value is 0;    -   G₁₀=1 if G is the base at position 10 on the sense strand,        otherwise its value is 0;    -   G₁₃=1 if G is the base at position 13 on the sense strand,        otherwise its value is 0;    -   G₁₉=1 if G is the base at position 19 of the sense strand,        otherwise if another base is present or the sense strand is only        18 base pairs in length, its value is 0;    -   U₁=1 if U is the base at position 1 on the sense strand,        otherwise its value is 0;    -   U₂=1 if U is the base at position 2 on the sense strand,        otherwise its value is 0;    -   U₃=1 if U is the base at position 3 on the sense strand,        otherwise its value is 0;    -   U₄=1 if U is the base at position 4 on the sense strand,        otherwise its value is 0;    -   U₇=1 if U is the base at position 7 on the sense strand,        otherwise its value is 0;    -   U₉=1 if U is the base at position 9 on the sense strand,        otherwise its value is 0;    -   U₁₀=1 if U is the base at position 10 on the sense strand,        otherwise its value is 0;    -   U₁₅=1 if U is the base at position 15 on the sense strand,        otherwise its value is 0;    -   U₁₆=1 if U is the base at position 16 on the sense strand,        otherwise its value is 0;    -   U₁₇=1 if U is the base at position 17 on the sense strand,        otherwise its value is 0;    -   U₁₈=1 if U is the base at position 18 on the sense strand,        otherwise its value is 0;    -   GC₁₅₋₁₉=the number of G and C bases within positions 15-19 of        the sense strand, or within positions 15-18 if the sense strand        is only 18 base pairs in length;    -   GC_(total)=the number of G and C bases in the sense strand;    -   Tm=100 if the siRNA oligo has the internal repeat longer then 4        base pairs, otherwise its value is 0; and    -   X=the number of times that the same nucleotide repeats four or        more times in a row.

The above formulas VIII, IX, and X, as well as formulas I-VII, providemethods for selecting siRNA in order to increase the efficiency of genesilencing. A subset of variables of any of the formulas may be used,though when fewer variables are used, the optimization hierarchy becomesless reliable.

With respect to the variables of the above-referenced formulas, a singleletter of A or C or G or U followed by a subscript refers to a binarycondition. The binary condition is that either the particular base ispresent at that particular position (wherein the value is “1”) or thebase is not present (wherein the value is “0”). Because position 19 isoptional, i.e., there might be only 18 base pairs, when there are only18 base pairs, any base with a subscript of 19 in the formulas abovewould have a zero value for that parameter. Before or after eachvariable is a number followed by *, which indicates that the value ofthe variable is to be multiplied or weighed by that number.

The numbers preceding the variables A, or G, or C, or U in FormulasVIII, IX, and X (or after the variables in Formula I-VII) weredetermined by comparing the difference in the frequency of individualbases at different positions in functional siRNA and total siRNA.Specifically, the frequency in which a given base was observed at aparticular position in functional groups was compared with the frequencythat that same base was observed in the total, randomly selected siRNAset. If the absolute value of the difference between the functional andtotal values was found to be greater than 6%, that parameter wasincluded in the equation. Thus, for instance, if the frequency offinding a “G” at position 13 (G13) is found to be 6% in a givenfunctional group, and the frequency of G₁₃ in the total population ofsiRNAs is 20%, the difference between the two values is 6%-20%=−14%. Asthe absolute value is greater than six (6), this factor (−14) isincluded in the equation. Thus, in Formula VIII, in cases where thesiRNA under study has a G in position 13, the accrued value is(−14)*(1)=−14. In contrast, when a base other than G is found atposition 13, the accrued value is (−14)*(0)=0.

When developing a means to optimize siRNAs, the inventors observed thata bias toward low internal thermodynamic stability of the duplex at the5′-antisense (AS) end is characteristic of naturally occurring miRNAprecursors. The inventors extended this observation to siRNAs for whichfunctionality had been assessed in tissue culture.

With respect to the parameter GC₁₅₋₁₉, a value of 0-5 will be ascribeddepending on the number of G or C bases at positions 15 to 19. If thereare only 18 base pairs, the value is between 0 and 4.

With respect to the criterion GC_(total) content, a number from 0-30will be ascribed, which correlates to the total number of G and Cnucleotides on the sense strand, excluding overhangs. Without wishing tobe bound by any one theory, it is postulated that the significance ofthe GC content (as well as AU content at positions 15-19, which is aparameter for formulas III-VII) relates to the easement of the unwindingof a double-stranded siRNA duplex. Duplex unwinding is believed to becrucial for siRNA functionality in vivo and overall low internalstability, especially low internal stability of the first unwound basepair is believed to be important to maintain sufficient processivity ofRISC complex-induced duplex unwinding. If the duplex has 19 base pairs,those at positions 15-19 on the sense strand will unwind first if themolecule exhibits a sufficiently low internal stability at thatposition. As persons skilled in the art are aware, RISC is a complex ofapproximately twelve proteins; Dicer is one, but not the only, helicasewithin this complex. Accordingly, although the GC parameters arebelieved to relate to activity with Dicer, they are also important foractivity with other RISC proteins.

The value of the parameter Tm is 0 when there are no internal repeatslonger than (or equal to) four base pairs present in the siRNA duplex;otherwise the value is 1. Thus for example, if the sequence ACGUACGU, orany other four nucleotide (or more) palindrome exists within thestructure, the value will be one (1). Alternatively if the structureACGGACG, or any other 3 nucleotide (or less) palindrome exists, thevalue will be zero (0).

The variable “X” refers to the number of times that the same nucleotideoccurs contiguously in a stretch of four or more units. If there are,for example, four contiguous As in one part of the sequence andelsewhere in the sequence four contiguous Cs, X=2. Further, if there aretwo separate contiguous stretches of four of the same nucleotides oreight or more of the same nucleotides in a row, then X=2. However, Xdoes not increase for five, six or seven contiguous nucleotides.

Again, when applying Formula VIII, Formula IX, or Formula X, to a givenmRNA, (the “target RNA” or “target molecule”), one may use a computerprogram to evaluate the criteria for every sequence of 18-30 base pairsor only sequences of a fixed length, e.g., 19 base pairs. Preferably thecomputer program is designed such that it provides a report ranking ofall of the potential siRNAs 18-30 base pairs, ranked according to whichsequences generate the highest value. A higher value refers to a moreefficient siRNA for a particular target gene. The computer program thatmay be used may be developed in any computer language that is known tobe useful for scoring nucleotide sequences, or it may be developed withthe assistance of commercially available product such as Microsoft'sproduct.net. Additionally, rather than run every sequence through oneand/or another formula, one may compare a subset of the sequences, whichmay be desirable if for example only a subset are available. Forinstance, it may be desirable to first perform a BLAST (Basic LocalAlignment Search Tool) search and to identify sequences that have nohomology to other targets. Alternatively, it may be desirable to scanthe sequence and to identify regions of moderate GC context, thenperform relevant calculations using one of the above-described formulason these regions. These calculations can be done manually or with theaid of a computer.

As with Formulas I-VII, either Formula VIII, Formula IX, or Formula Xmay be used for a given mRNA target sequence. However, it is possiblethat according to one or the other formula more than one siRNA will havethe same value. Accordingly, it is beneficial to have a second formulaby which to differentiate sequences. Formulas IX and X were derived in asimilar fashion as Formula VIII, yet used a larger data set and thusyields sequences with higher statistical correlations to highlyfunctional duplexes. The sequence that has the highest value ascribed toit may be referred to as a “first optimized duplex.” The sequence thathas the second highest value ascribed to it may be referred to as a“second optimized duplex.” Similarly, the sequences that have the thirdand fourth highest values ascribed to them may be referred to as a thirdoptimized duplex and a fourth optimized duplex, respectively. When morethan one sequence has the same value, each of them may, for example, bereferred to as first optimized duplex sequences or co-first optimizedduplexes. Formula X is similar to Formula IX, yet uses a greater numbersof variables and for that reason, identifies sequences on the basis ofslightly different criteria.

It should also be noted that the output of a particular algorithm willdepend on several of variables including: (1) the size of the database(s) being analyzed by the algorithm, and (2) the number andstringency of the parameters being applied to screen each sequence.Thus, for example, in U.S. patent application Ser. No. 10/714,333,entitled “Functional and Hyperfunctional siRNA,” filed Nov. 14, 2003,Formula VIII was applied to the known human genome (ncbi refseqdatabase) through Entrez (efetch). As a result of these procedures,roughly 1.6 million siRNA sequences were identified. Application ofFormula VIII to the same database in March of 2004 yielded roughly 2.2million sequences, a difference of approximately 600,000 sequencesresulting from the growth of the database over the course of the monthsthat span this period of time. Application of other formulas (e.g.,Formula X) that change the emphasis of, include, or eliminate differentvariables can yield unequal numbers of siRNAs. Alternatively, in caseswhere application of one formula to one or more genes fails to yieldsufficient numbers of siRNAs with scores that would be indicative ofstrong silencing, said genes can be reassessed with a second algorithmthat is, for instance, less stringent.

siRNA sequences identified using Formula VIII and Formula X (minussequences generated by Formula VIII) are contained within the enclosedcompact disks. The data included on the enclosed compact disks isdescribed more fully below. The sequences identified by Formula VIII andFormula X that are disclosed in the compacts disks may be used in genesilencing applications.

It should be noted that for Formulas VIII, IX, and X all of theaforementioned criteria are identified as positions on the sense strandwhen oriented in the 5′ to 3′ direction as they are identified inconnection with Formulas I-VII unless otherwise specified.

Formulas I-X, may be used to select or to evaluate one, or more thanone, siRNA in order to optimize silencing. Preferably, at least twooptimized siRNAs that have been selected according to at least one ofthese formulas are used to silence a gene, more preferably at leastthree and most preferably at least four. The siRNAs may be usedindividually or together in a pool or kit. Further, they may be appliedto a cell simultaneously or separately. Preferably, the at least twosiRNAs are applied simultaneously. Pools are particularly beneficial formany research applications. However, for therapeutics, it may be moredesirable to employ a single hyperfunctional siRNA as describedelsewhere in this application.

When planning to conduct gene silencing, and it is necessary to choosebetween two or more siRNAs, one should do so by comparing the relativevalues when the siRNA are subjected to one of the formulas above. Ingeneral a higher scored siRNA should be used.

Useful applications include, but are not limited to, target validation,gene functional analysis, research and drug discovery, gene therapy andtherapeutics. Methods for using siRNA in these applications are wellknown to persons of skill in the art.

Because the ability of siRNA to function is dependent on the sequence ofthe RNA and not the species into which it is introduced, the presentinvention is applicable across a broad range of species, including butnot limited to all mammalian species, such as humans, dogs, horses,cats, cows, mice, hamsters, chimpanzees and gorillas, as well as otherspecies and organisms such as bacteria, viruses, insects, plants and C.elegans.

The present invention is also applicable for use for silencing a broadrange of genes, including but not limited to the roughly 45,000 genes ofa human genome, and has particular relevance in cases where those genesare associated with diseases such as diabetes, Alzheimer's, cancer, aswell as all genes in the genomes of the aforementioned organisms.

The siRNA selected according to the aforementioned criteria or one ofthe aforementioned algorithms are also, for example, useful in thesimultaneous screening and functional analysis of multiple genes andgene families using high throughput strategies, as well as in directgene suppression or silencing.

Development of the Algorithms

To identify siRNA sequence features that promote functionality and toquantify the importance of certain currently accepted conventionalfactors—such as G/C content and target site accessibility—the inventorssynthesized an siRNA panel consisting of 270 siRNAs targeting threegenes, Human Cyclophilin, Firefly Luciferase, and Human DBI. In allthree cases, siRNAs were directed against specific regions of each gene.For Human Cyclophilin and Firefly Luciferase, ninety siRNAs weredirected against a 199 bp segment of each respective mRNA. For DBI, 90siRNAs were directed against a smaller, 109 base pair region of themRNA. The sequences to which the siRNAs were directed are providedbelow.

It should be noted that in certain sequences, “t” is present. This isbecause many databases contain information in this manner. However, thet denotes a uracil residue in mRNA and siRNA. Any algorithm will, unlessotherwise specified, process a t in a sequence as a u.

Human cyclophilin: 193-390 M60857 gttccaaaaacagtggataattttgtggccttagctSEQ. ID NO. 29 acaggagagaaaggatttggctacaaaaacagcaaattccatcgtgtaatcaaggacttcatgatccagggcggagacttcaccaggggagatggcacaggaggaaagagcatctacggtgagcgcttccccgatgagaacttc aaactgaagcactacgggcctggctggg:

Firefly luciferase: 1434-1631, U47298 (pGL3, Promega)tgaacttcccgccgccgttgttgttttggagcacgg SEQ. ID NO. 30aaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaactcgacgcaagaaaaatcagagagat cctcataaaggccaagaagg:

DBI, NM_(—)020548 (202-310) (Every Position)acgggcaaggccaagtgggatgcctggaatgagc SEQ. ID NO. 0031tgaaagggacttccaaggaagatgccatgaaagc ttacatcaacaaagtagaagagctaaagaaaaaatacggg:A list of the siRNAs appears in Table III (see Examples Section, ExampleII)

The set of duplexes was analyzed to identify correlations between siRNAfunctionality and other biophysical or thermodynamic properties. Whenthe siRNA panel was analyzed in functional and non-functional subgroups,certain nucleotides were much more abundant at certain positions infunctional or non-functional groups. More specifically, the frequency ofeach nucleotide at each position in highly functional siRNA duplexes wascompared with that of nonfunctional duplexes in order to assess thepreference for or against any given nucleotide at every position. Theseanalyses were used to determine important criteria to be included in thesiRNA algorithms (Formulas VIII, IX, and X).

The data set was also analyzed for distinguishing biophysical propertiesof siRNAs in the functional group, such as optimal percent of GCcontent, propensity for internal structures and regional thermodynamicstability. Of the presented criteria, several are involved in duplexrecognition, RISC activation/duplex unwinding, and target cleavagecatalysis.

The original data set that was the source of the statistically derivedcriteria is shown in FIG. 2. Additionally, this figure shows that randomselection yields siRNA duplexes with unpredictable and widely varyingsilencing potencies as measured in tissue culture using HEK293 cells. Inthe figure, duplexes are plotted such that each x-axis tick-markrepresents an individual siRNA, with each subsequent siRNA differing intarget position by two nucleotides for Human Cyclophilin B and FireflyLuciferase, and by one nucleotide for Human DBI. Furthermore, the y-axisdenotes the level of target expression remaining after transfection ofthe duplex into cells and subsequent silencing of the target.

siRNA identified and optimized in this document work equally well in awide range of cell types. FIG. 3 a shows the evaluation of thirty siRNAstargeting the DBI gene in three cell lines derived from differenttissues. Each DBI siRNA displays very similar functionality in HEK293(ATCC, CRL-1573, human embryonic kidney), HeLa (ATCC, CCL-2, cervicalepithelial adenocarcinoma) and DU145 (HTB-81, prostate) cells asdeterimined by the B-DNA assay. Thus, siRNA functionality is determinedby the primary sequence of the siRNA and not by the intracellularenvironment. Additionally, it should be noted that although the presentinvention provides for a determination of the functionality of siRNA fora given target, the same siRNA may silence more than one gene. Forexample, the complementary sequence of the silencing siRNA may bepresent in more than one gene. Accordingly, in these circumstances, itmay be desirable not to use the siRNA with highest SMARTscore™. In suchcircumstances, it may be desirable to use the siRNA with the nexthighest SMARTscore™.

To determine the relevance of G/C content in siRNA function, the G/Ccontent of each duplex in the panel was calculated and the functionalclasses of siRNAs (<F50,≧F50, ≧F80,≧F95 where F refers to the percentgene silencing) were sorted accordingly. The majority of thehighly-functional siRNAs (≧F95) fell within the G/C content range of36-52% (FIG. 3B). Twice as many non-functional (<F50) duplexes fellwithin the high G/C content groups (>57% GC content) compared to the36%-52% group. The group with extremely low GC content (26% or less)contained a higher proportion of non-functional siRNAs and nohighly-functional siRNAs. The G/C content range of 30%-52% was thereforeselected as Criterion I for siRNA functionality, consistent with theobservation that a G/C range 30%-70% promotes efficient RNAi targeting.Application of this criterion alone provided only a marginal increase inthe probability of selecting functional siRNAs from the panel: selectionof F50 and F95 siRNAs was improved by 3.6% and 2.2%, respectively. ThesiRNA panel presented here permitted a more systematic analysis andquantification of the importance of this criterion than that usedpreviously.

A relative measure of local internal stability is the A/U base pair (bp)content; therefore, the frequency of A/U bp was determined for each ofthe five terminal positions of the duplex (5′ sense (S)/5′ antisense(AS)) of all siRNAs in the panel. Duplexes were then categorized by thenumber of A/U bp in positions 1-5 and 15-19 of the sense strand. Thethermodynamic flexibility of the duplex 5′-end (positions 1-5; S) didnot appear to correlate appreciably with silencing potency, while thatof the 3′-end (positions 15-19; S) correlated with efficient silencing.No duplexes lacking A/U bp in positions 15-19 were functional. Thepresence of one A/U bp in this region conferred some degree offunctionality, but the presence of three or more A/Us was preferable andtherefore defined as Criterion II. When applied to the test panel, onlya marginal increase in the probability of functional siRNA selection wasachieved: a 1.8% and 2.3% increase for F50 and F95 duplexes,respectively (Table IV).

The complementary strands of siRNAs that contain internal repeats orpalindromes may form internal fold-back structures. These hairpin-likestructures exist in equilibrium with the duplexed form effectivelyreducing the concentration of functional duplexes. The propensity toform internal hairpins and their relative stability can be estimated bypredicted melting temperatures. High Tm reflects a tendency to formhairpin structures. Lower Tm values indicate a lesser tendency to formhairpins. When the functional classes of siRNAs were sorted by T_(m)(FIG. 3 c), the following trends were identified: duplexes lackingstable internal repeats were the most potent silencers (no F95 duplexwith predicted hairpin structure T_(m)>60° C.). In contrast, about 60%of the duplexes in the groups having internal hairpins with calculatedT_(m) values less than 20° C. were F80. Thus, the stability of internalrepeats is inversely proportional to the silencing effect and definesCriterion III (predicted hairpin structure T_(m)≦20° C.).

Sequence-Based Determinants of siRNA Functionality

When the siRNA panel was sorted into functional and non-functionalgroups, the frequency of a specific nucleotide at each position in afunctional siRNA duplex was compared with that of a nonfunctional duplexin order to assess the preference for or against a certain nucleotide.FIG. 4 shows the results of these queries and the subsequent resortingof the data set (from FIG. 2). The data is separated into two sets:those duplexes that meet the criteria, a specific nucleotide in acertain position—grouped on the left (Selected) and those that donot—grouped on the right (Eliminated). The duplexes are further sortedfrom most functional to least functional with the y-axis of FIG. 4 a-erepresenting the % expression i.e., the amount of silencing that iselicited by the duplex (Note: each position on the X-axis represents adifferent duplex). Statistical analysis revealed correlations betweensilencing and several sequence-related properties of siRNAs. FIG. 4 andTable IV show quantitative analysis for the following fivesequence-related properties of siRNA: (A) an A at position 19 of thesense strand; (B) an A at position 3 of the sense strand; (C) a U atposition 10 of the sense strand; (D) a base other than G at position 13of the sense strand; and (E) a base other than C at position 19 of thesense strand.

When the siRNAs in the panel were evaluated for the presence of an A atposition 19 of the sense strand, the percentage of non-functionalduplexes decreased from 20% to 11.8%, and the percentage of F95 duplexesincreased from 21.7% to 29.4% (Table IV). Thus, the presence of an A inthis position defined Criterion IV.

Another sequence-related property correlated with silencing was thepresence of an A in position 3 of the sense strand (FIG. 4 b). Of thesiRNAs with A3, 34.4% were F95, compared with 21.7% randomly selectedsiRNAs. The presence of a U base in position 10 of the sense strandexhibited an even greater impact (FIG. 4 c). Of the duplexes in thisgroup, 41.7% were F95. These properties became criteria V and VI,respectively.

Two negative sequence-related criteria that were identified also appearon FIG. 4. The absence of a G at position 13 of the sense strand,conferred a marginal increase in selecting functional duplexes (FIG. 4d). Similarly, lack of a C at position 19 of the sense strand alsocorrelated with functionality (FIG. 4 e). Thus, among functionalduplexes, position 19 was most likely occupied by A, and rarely occupiedby C. These rules were defined as criteria VII and VIII, respectively.

Application of each criterion individually provided marginal butstatistically significant increases in the probability of selecting apotent siRNA. Although the results were informative, the inventorssought to maximize potency and therefore consider multiple criteria orparameters. Optimization is particularly important when developingtherapeutics. Interestingly, the probability of selecting a functionalsiRNA based on each thermodynamic criteria was 2%-4% higher than random,but 4%-8% higher for the sequence-related determinates. Presumably,these sequence-related increases reflect the complexity of the RNAimechanism and the multitude of protein-RNA interactions that areinvolved in RNAi-mediated silencing. TABLE IV Improvement Criterion %Functional over Random I. 30%-52% G/C content <F50 16.4% −3.6%   ≧F5083.6% 3.6% ≧F80 60.4% 4.3% ≧F95 23.9% 2.2% II. At least 3 A/U bases atpositions <F50 18.2% −1.8%   15-19 of the sense strand ≧F50 81.8% 1.8%≧F80 59.7% 3.6% ≧F95 24.0% 2.3% III. Absence of internal repeats, <F5016.7% −3.3%   as measured by T_(m) of ≧F50 83.3% 3.3% secondarystructure ≦20° C. ≧F80 61.1% 5.0% ≧F95 24.6% 2.9% IV. An A base atposition 19 <F50 11.8% −8.2%   of the sense strand ≧F50 88.2% 8.2% ≧F8075.0% 18.9%  ≧F95 29.4% 7.7% V. An A base at position 3 <F50 17.2%−2.8%   of the sense strand ≧F50 82.8% 2.8% ≧F80 62.5% 6.4% ≧F95 34.4%12.7%  VI. A U base at position 10 <F50 13.9% −6.1%   of the sensestrand ≧F50 86.1% 6.1% ≧F80 69.4% 13.3%  ≧F95 41.7%  20% VII. A baseother than C at <F50 18.8% −1.2%   position 19 of the sense strand ≧F5081.2% 1.2% ≧F80 59.7% 3.6% ≧F95 24.2% 2.5% VIII. A base other than G at<F50 15.2% −4.8%   position 13 of the sense strand ≧F50 84.8% 4.8% ≧F8061.4% 5.3% ≧F95 26.5% 4.8%The siRNA Selection Algorithm

In an effort to improve selection further, all identified criteria,including but not limited to those listed in Table IV were combined intothe algorithms embodied in Formula VIII, Formula IX, and Formula X. EachsiRNA was then assigned a score (referred to as a SMARTscore™) accordingto the values derived from the formulas. Duplexes that scored higherthan 0 or −20 (unadjusted), for Formulas VIII and IX, respectively,effectively selected a set of functional siRNAs and excluded allnon-functional siRNAs. Conversely, all duplexes scoring lower than 0 and−20 (minus 20) according to formulas VIII and IX, respectively,contained some functional siRNAs but included all non-functional siRNAs.A graphical representation of this selection is shown in FIG. 5. Itshould be noted that the scores derived from the algorithm can also beprovided as “adjusted” scores. To convert Formula VIII unadjusted scoresinto adjusted scores it is necessary to use the following equation:(160+unadjusted score)/2.25When this takes place, an unadjusted score of “0” (zero) is converted to75. Similarly, unadjusted scores for Formula X can be converted toadjusted scores. In this instance, the following equation is applied:(228+unadjusted score)/3.56When these manipulations take place, an unadjusted score of 38 isconverted to an adjusted score of 75.

The methods for obtaining the seven criteria embodied in Table IV areillustrative of the results of the process used to develop theinformation for Formulas VIII, IX, and X. Thus similar techniques wereused to establish the other variables and their multipliers. Asdescribed above, basic statistical methods were use to determine therelative values for these multipliers.

To determine the value for “Improvement over Random” the difference inthe frequency of a given attribute (e.g., GC content, base preference)at a particular position is determined between individual functionalgroups (e.g., <F50) and the total siRNA population studied (e.g., 270siRNA molecules selected randomly). Thus, for instance, in Criterion I(30%-52% GC content) members of the <F50 group were observed to have GCcontents between 30-52% in 16.4% of the cases. In contrast, the totalgroup of 270 siRNAs had GC contents in this range, 20% of the time. Thusfor this particular attribute, there is a small negative correlationbetween 30%-52% GC content and this functional group (i.e.,16.4%-20%=−3.6%). Similarly, for Criterion VI, (a “U” at position 10 ofthe sense strand), the >F95 group contained a “U” at this position 41.7%of the time. In contrast, the total group of 270 siRNAs had a “U” atthis position 21.7% of the time, thus the improvement over random iscalculated to be 20% (or 41.7%-21.7%).

Identifying the Average Internal Stability Profile of Strong siRNA

In order to identify an internal stability profile that ischaracteristic of strong siRNA, 270 different siRNAs derived from thecyclophilin B, the diazepam binding inhibitor (DBI), and the luciferasegene were individually transfected into HEK293 cells and tested fortheir ability to induce RNAi of the respective gene. Based on theirperformance in the in vivo assay, the sequences were then subdividedinto three groups, (i) >95% silencing; (ii) 80-95% silencing; and (iii)less than 50% silencing. Sequences exhibiting 51-84% silencing wereeliminated from further consideration to reduce the difficulties inidentifying relevant thermodynamic patterns.

Following the division of siRNA into three groups, a statisticalanalysis was performed on each member of each group to determine theaverage internal stability profile (AISP) of the siRNA. To accomplishthis the Oligo 5.0 Primer Analysis Software and other relatedstatistical packages (e.g., Excel) were exploited to determine theinternal stability of pentamers using the nearest neighbor methoddescribed by Freier et al., (1986) Improved free-energy parameters forpredictions of RNA duplex stability, Proc Natl. Acad. Sci. U.S.A.83(24): 9373-7. Values for each group at each position were thenaveraged, and the resulting data were graphed on a linear coordinatesystem with the Y-axis expressing the ΔG (free energy) values inkcal/mole and the X-axis identifying the position of the base relativeto the 5′ end.

The results of the analysis identified multiple key regions in siRNAmolecules that were critical for successful gene silencing. At the3′-most end of the sense strand (5′antisense), highly functional siRNA(>95% gene silencing, see FIG. 6 a, >F95) have a low internal stability(AISP of position 19=˜7.6 kcal/mol). In contrast low-efficiency siRNA(i.e., those exhibiting less than 50% silencing, <F50) display adistinctly different profile, having high ΔG values (˜−8.4 kcal/mol) forthe same position. Moving in a 5′ (sense strand) direction, the internalstability of highly efficient siRNA rises (position 12=˜−8.3 kcal/mole)and then drops again (position 7=˜−7.7 kcal/mol) before leveling off ata value of approximately −8.1 kcal/mol for the 5′ terminus. siRNA withpoor silencing capabilities show a distinctly different profile. Whilethe AISP value at position 12 is nearly identical with that of strongsiRNAs, the values at positions 7 and 8 rise considerably, peaking at ahigh of ˜−9.0 kcal/mol. In addition, at the 5′ end of the molecule theAISP profile of strong and weak siRNA differ dramatically. Unlike therelatively strong values exhibited by siRNA in the >95% silencing group,siRNAs that exhibit poor silencing activity have weak AISP values (−7.6,−7.5, and −7.5 kcal/mol for positions 1, 2 and 3 respectively).

Overall the profiles of both strong and weak siRNAs form distinctsinusoidal shapes that are roughly 180° out-of-phase with each other.While these thermodynamic descriptions define the archetypal profile ofa strong siRNA, it will likely be the case that neither the ΔG valuesgiven for key positions in the profile or the absolute position of theprofile along the Y-axis (i.e., the ΔG-axis) are absolutes. Profilesthat are shifted upward or downward (i.e., having on an average, higheror lower values at every position) but retain the relative shape andposition of the profile along the X-axis can be foreseen as beingequally effective as the model profile described here. Moreover, it islikely that siRNA that have strong or even stronger gene-specificsilencing effects might have exaggerated ΔG values (either higher orlower) at key positions. Thus, for instance, it is possible that the5′-most position of the sense strand (position 19) could have ΔG valuesof 7.4 kcal/mol or lower and still be a strong siRNA if, for instance, aG-C→G-T/U mismatch were substituted at position 19 and altered duplexstability. Similarly, position 12 and position 7 could have values above8.3 kcal/mol and below 7.7 kcal/mole, respectively, without abating thesilencing effectiveness of the molecule. Thus, for instance, at position12, a stabilizing chemical modification (e.g., a chemical modificationof the 2′ position of the sugar backbone) could be added that increasesthe average internal stability at that position. Similarly, at position7, mismatches similar to those described previously could be introducedthat would lower the ΔG values at that position.

Lastly, it is important to note that while functional and non-functionalsiRNA were originally defined as those molecules having specificsilencing properties, both broader or more limiting parameters can beused to define these molecules. As used herein, unless otherwisespecified, “non-functional siRNA” are defined as those siRNA that induceless than 50% (<50%) target silencing, “semi-functional siRNA” induce50-79% target silencing, “functional siRNA” are molecules that induce80-95% gene silencing, and “highly-functional siRNA” are molecules thatinduce great than 95% gene silencing. These definitions are not intendedto be rigid and can vary depending upon the design and needs of theapplication. For instance, it is possible that a researcher attemptingto map a gene to a chromosome using a functional assay, may identify ansiRNA that reduces gene activity by only 30%. While this level of genesilencing may be “non-functional” for, e.g., therapeutic needs, it issufficient for gene mapping purposes and is, under these uses andconditions, “functional.” For these reasons, functional siRNA can bedefined as those molecules having greater than 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% silencing capabilities at 100 nM transfectionconditions. Similarly, depending upon the needs of the study and/orapplication, non-functional and semi-functional siRNA can be defined ashaving different parameters. For instance, semi-functional siRNA can bedefined as being those molecules that induce 20%, 30%, 40%, 50%, 60%, or70% silencing at 100 nM transfection conditions. Similarly,non-functional siRNA can be defined as being those molecules thatsilence gene expression by less than 70%, 60%, 50%, 40%, 30%, or less.Nonetheless, unless otherwise stated, the descriptions stated in the“Definitions” section of this text should be applied.

Functional attributes can be assigned to each of the key positions inthe AISP of strong siRNA. The low 5′ (sense strand) AISP values ofstrong siRNAs may be necessary for determining which end of the moleculeenters the RISC complex. In contrast, the high and low AISP valuesobserved in the central regions of the molecule may be critical forsiRNA-target mRNA interactions and product release, respectively.

If the AISP values described above accurately define the thermodynamicparameters of strong siRNA, it would be expected that similar patternswould be observed in strong siRNA isolated from nature. Natural siRNAsexist in a harsh, RNase-rich environment and it can be hypothesized thatonly those siRNA that exhibit heightened affinity for RISC (i.e., siRNAthat exhibit an average internal stability profile similar to thoseobserved in strong siRNA) would survive in an intracellular environment.This hypothesis was tested using GFP-specific siRNA isolated from N.benthamiana. Llave et al. (2002) Endogenous and Silencing-AssociatedSmall RNAs in Plants, The Plant Cell 14, 1605-1619, introduced longdouble-stranded GFP-encoding RNA into plants and subsequentlyre-isolated GFP-specific siRNA from the tissues. The AISP of fifty-nineof these GFP-siRNA were determined, averaged, and subsequently plottedalongside the AISP profile obtained from the cyclophilinB/DBI/luciferase siRNA having >90% silencing properties (FIG. 6 b).Comparison of the two groups show that profiles are nearly identical.This finding validates the information provided by the internalstability profiles and demonstrates that: (1) the profile identified byanalysis of the cyclophilin B/DBI/luciferase siRNAs are not genespecific; and (2) AISP values can be used to search for strong siRNAs ina variety of species.

Both chemical modifications and base-pair mismatches can be incorporatedinto siRNA to alter the duplex's AISP and functionality. For instance,introduction of mismatches at positions 1 or 2 of the sense stranddestabilized the 5′ end of the sense strand and increases thefunctionality of the molecule (see Luc, FIG. 7). Similarly, addition of2′-O-methyl groups to positions 1 and 2 of the sense strand can alsoalter the AISP and (as a result) increase both the functionality of themolecule and eliminate off-target effects that results from sense strandhomology with the unrelated targets (FIGS. 8 a, 8 b).

Rationale for Criteria in a Biological Context

The fate of siRNA in the RNAi pathway may be described in 5 major steps:(1) duplex recognition and pre-RISC complex formation; (2) ATP-dependentduplex unwinding/strand selection and RISC activation; (3) mRNA targetidentification; (4) mRNA cleavage, and (5) product release (FIG. 1).Given the level of nucleic acid-protein interactions at each step, siRNAfunctionality is likely influenced by specific biophysical and molecularproperties that promote efficient interactions within the context of themulti-component complexes. Indeed, the systematic analysis of the siRNAtest set identified multiple factors that correlate well withfunctionality. When combined into a single algorithm, they proved to bevery effective in selecting active siRNAs.

The factors described here may also be predictive of key functionalassociations important for each step in RNAi. For example, the potentialformation of internal hairpin structures correlated negatively withsiRNA functionality. Complementary strands with stable internal repeatsare more likely to exist as stable hairpins thus decreasing theeffective concentration of the functional duplex form. This suggeststhat the duplex is the preferred conformation for initial pre-RISCassociation. Indeed, although single complementary strands can inducegene silencing, the effective concentration required is at least twoorders of magnitude higher than that of the duplex form.

siRNA-pre-RISC complex formation is followed by an ATP-dependent duplexunwinding step and “activation” of the RISC. The siRNA functionality wasshown to correlate with overall low internal stability of the duplex andlow internal stability of the 3′ sense end (or differential internalstability of the 3′ sense compare to the 5′ sense strand), which mayreflect strand selection and entry into the RISC. Overall duplexstability and low internal stability at the 3′ end of the sense strandwere also correlated with siRNA functionality. Interestingly, siRNAswith very high and very low overall stability profiles correlatestrongly with non-functional duplexes. One interpretation is that highinternal stability prevents efficient unwinding while very low stabilityreduces siRNA target affinity and subsequent mRNA cleavage by the RISC.

Several criteria describe base preferences at specific positions of thesense strand and are even more intriguing when considering theirpotential mechanistic roles in target recognition and mRNA cleavage.Base preferences for A at position 19 of the sense strand but not C, areparticularly interesting because they reflect the same base preferencesobserved for naturally occurring miRNA precursors. That is, among thereported miRNA precursor sequences 75% contain a U at position 1 whichcorresponds to an A in position 19 of the sense strand of siRNAs, whileG was under-represented in this same position for miRNA precursors.These observations support the hypothesis that both miRNA precursors andsiRNA duplexes are processed by very similar if not identical proteinmachinery. The functional interpretation of the predominance of a U/Abase pair is that it promotes flexibility at the 5′antisense ends ofboth siRNA duplexes and miRNA precursors and facilitates efficientunwinding and selective strand entrance into an activated RISC.

Among the criteria associated with base preferences that are likely toinfluence mRNA cleavage or possibly product release, the preference forU at position 10 of the sense strand exhibited the greatest impact,enhancing the probability of selecting an F80 sequence by 13.3%.Activated RISC preferentially cleaves target mRNA between nucleotides 10and 11 relative to the 5′ end of the complementary targeting strand.Therefore, it may be that U, the preferred base for mostendoribonucleases, at this position supports more efficient cleavage.Alternatively, a U/A bp between the targeting siRNA strand and itscognate target mRNA may create an optimal conformation for theRISC-associated “slicing” activity.

Post Algorithm Filters

According to another embodiment, the output of any one of the formulaspreviously listed can be filtered to remove or select for siRNAscontaining undesirable or desirable motifs or properties, respectively.In one example, sequences identified by any of the formulas can befiltered to remove any and all sequences that induce toxicity orcellular stress. Introduction of an siRNA containing a toxic motif intoa cell can induce cellular stress and/or cell death (apoptosis) which inturn can mislead researchers into associating a particular (e.g.,nonessential) gene with, e.g., an essential function. Alternatively,sequences generated by any of the before mentioned formulas can befiltered to identify and retain duplexes that contain toxic motifs. Suchduplexes may be valuable from a variety of perspectives including, forinstance, uses as therapeutic molecules. A variety of toxic motifs existand can exert their influence on the cell through RNAi and non-RNAipathways. Examples of toxic motifs are explained more fully in commonlyassigned U.S. Provisional Patent Application Ser. No. 60/538,874,entitled “Identification of Toxic Sequences,” filed Jan. 23, 2004.Briefly, toxic motifs include A/G UUU A/G/U, G/C AAA G/C, and GCCA, or acomplement of any of the foregoing.

In another instance, sequences identified by any of the before mentionedformulas can be filtered to identify duplexes that contain motifs (orgeneral properties) that provide serum stability or induce seruminstability. In one envisioned application of siRNA as therapeuticmolecules, duplexes targeting disease-associated genes will beintroduced into patients intravenously. As the half-life of single anddouble stranded RNA in serum is short, post-algorithm filters designedto select molecules that contain motifs that enhance duplex stability inthe presence of serum and/or (conversely) eliminate duplexes thatcontain motifs that destabilize siRNA in the presence of serum, would bebeneficial.

In another instance, sequences identified by any of the before mentionedformulas can be filtered to identify duplexes that are hyperfunctional.Hyperfunctional sequences are defined as those sequences that (1) inducegreater than 95% silencing of a specific target when they aretransfected at subnanomolar concentrations (i.e., less than onenanomolar); and/or (2) induce functional (or better) levels of silencingfor greater than 96 hours. Filters that identify hyperfunctionalmolecules can vary widely. In one example, the top ten, twenty, thirty,or forty siRNA can be assessed for the ability to silence a given targetat, e.g., concentrations of 1 nM and 0.5 nM to identify hyperfunctionalmolecules.

Pooling

According to another embodiment, the present invention provides a poolof at least two siRNAs, preferably in the form of a kit or therapeuticreagent, wherein one strand of each of the siRNAs, the sense strandcomprises a sequence that is substantially similar to a sequence withina target mRNA. The opposite strand, the antisense strand, willpreferably comprise a sequence that is substantially complementary tothat of the target mRNA. More preferably, one strand of each siRNA willcomprise a sequence that is identical to a sequence that is contained inthe target mRNA. Most preferably, each siRNA will be 19 base pairs inlength, and one strand of each of the siRNAs will be 100% complementaryto a portion of the target mRNA.

By increasing the number of siRNAs directed to a particular target usinga pool or kit, one is able both to increase the likelihood that at leastone siRNA with satisfactory functionality will be included, as well asto benefit from additive or synergistic effects. Further, when two ormore siRNAs directed against a single gene do not have satisfactorylevels of functionality alone, if combined, they may satisfactorilypromote degradation of the target messenger RNA and successfully inhibittranslation. By including multiple siRNAs in the system, not only is theprobability of silencing increased, but the economics of operation arealso improved when compared to adding different siRNAs sequentially.This effect is contrary to the conventional wisdom that the concurrentuse of multiple siRNA will negatively impact gene silencing (e.g.,Holen, T. et al. (2003) “Similar behavior of single strand and doublestrand siRNAs suggests they act through a common RNAi pathway.” NAR 31:2401-21407).

In fact, when two siRNAs were pooled together, 54% of the pools of twosiRNAs induced more than 95% gene silencing. Thus, a 2.5-fold increasein the percentage of functionality was achieved by randomly combiningtwo siRNAs. Further, over 84% of pools containing two siRNAs inducedmore than 80% gene silencing.

More preferably, the kit is comprised of at least three siRNAs, whereinone strand of each siRNA comprises a sequence that is substantiallysimilar to a sequence of the target mRNA and the other strand comprisesa sequence that is substantially complementary to the region of thetarget mRNA. As with the kit that comprises at least two siRNAs, morepreferably one strand will comprise a sequence that is identical to asequence that is contained in the mRNA and another strand that is 100%complementary to a sequence that is contained in the mRNA. Duringexperiments, when three siRNAs were combined together, 60% of the poolsinduced more than 95% gene silencing and 92% of the pools induced morethan 80% gene silencing.

Further, even more preferably, the kit is comprised of at least foursiRNAs, wherein one strand of each siRNA comprises a sequence that issubstantially similar to a region of the sequence of the target mRNA,and the other strand comprises a sequence that is substantiallycomplementary to the region of the target mRNA. As with the kit or poolthat comprises at least two siRNAs, more preferably one strand of eachof the siRNA duplexes will comprise a sequence that is identical to asequence that is contained in the mRNA, and another strand that is 100%complementary to a sequence that is contained in the mRNA.

Additionally, kits and pools with at least five, at least six, and atleast seven siRNAs may also be useful with the present invention. Forexample, pools of five siRNA induced 95% gene silencing with 77%probability and 80% silencing with 98.8% probability. Thus, pooling ofsiRNAs together can result in the creation of a target-specificsilencing reagent with almost a 99% probability of being functional. Thefact that such high levels of success are achievable using such pools ofsiRNA, enables one to dispense with costly and time-consumingtarget-specific validation procedures.

For this embodiment, as well as the other aforementioned embodiments,each of the siRNAs within a pool will preferably comprise 18-30 basepairs, more preferably 18-25 base pairs, and most preferably 19 basepairs. Within each siRNA, preferably at least 18 contiguous bases of theantisense strand will be 100% complementary to the target mRNA. Morepreferably, at least 19 contiguous bases of the antisense strand will be100% complementary to the target mRNA. Additionally, there may beoverhangs on either the sense strand or the antisense strand, and theseoverhangs may be at either the 5′ end or the 3′ end of either of thestrands, for example there may be one or more overhangs of 1-6 bases.When overhangs are present, they are not included in the calculation ofthe number of base pairs. The two nucleotide 3′ overhangs mimic naturalsiRNAs and are commonly used but are not essential. Preferably, theoverhangs should consist of two nucleotides, most often dTdT or UU atthe 3′ end of the sense and antisense strand that are not complementaryto the target sequence. The siRNAs may be produced by any method that isnow known or that comes to be known for synthesizing double stranded RNAthat one skilled in the art would appreciate would be useful in thepresent invention. Preferably, the siRNAs will be produced byDharmacon's proprietary ACE® technology. However, other methods forsynthesizing siRNAs are well known to persons skilled in the art andinclude, but are not limited to, any chemical synthesis of RNAoligonucleotides, ligation of shorter oligonucleotides, in vitrotranscription of RNA oligonucleotides, the use of vectors for expressionwithin cells, recombinant Dicer products and PCR products.

The siRNA duplexes within the aforementioned pools of siRNAs maycorrespond to overlapping sequences within a particular mRNA, ornon-overlapping sequences of the mRNA. However, preferably theycorrespond to non-overlapping sequences. Further, each siRNA may beselected randomly, or one or more of the siRNA may be selected accordingto the criteria discussed above for maximizing the effectiveness ofsiRNA.

Included in the definition of siRNAs are siRNAs that contain substitutedand/or labeled nucleotides that may, for example, be labeled byradioactivity, fluorescence or mass. The most common substitutions areat the 2′ position of the ribose sugar, where moieties such as H(hydrogen) F, NH₃, OCH₃ and other O- alkyl, alkenyl, alkynyl, andorthoesters, may be substituted, or in the phosphorous backbone, wheresulfur, amines or hydrocarbons may be substituted for the bridging ofnon-bridging atoms in the phosphodiester bond. Examples of modifiedsiRNAs are explained more fully in commonly assigned U.S. patentapplication Ser. No. 10/613,077, filed Jul. 1, 2003.

Additionally, as noted above, the cell type into which the siRNA isintroduced may affect the ability of the siRNA to enter the cell;however, it does not appear to affect the ability of the siRNA tofunction once it enters the cell. Methods for introducingdouble-stranded RNA into various cell types are well known to personsskilled in the art.

As persons skilled in the art are aware, in certain species, thepresence of proteins such as RdRP, the RNA-dependent RNA polymerase, maycatalytically enhance the activity of the siRNA. For example, RdRPpropagates the RNAi effect in C. elegans and other non-mammalianorganisms. In fact, in organisms that contain these proteins, the siRNAmay be inherited. Two other proteins that are well studied and known tobe a part of the machinery are members of the Argonaute family andDicer, as well as their homologues. There is also initial evidence thatthe RISC complex might be associated with the ribosome so the moreefficiently translated mRNAs will be more susceptible to silencing thanothers.

Another very important factor in the efficacy of siRNA is mRNAlocalization. In general, only cytoplasmic mRNAs are considered to beaccessible to RNAi to any appreciable degree. However, appropriatelydesigned siRNAs, for example, siRNAs modified with internucleotidelinkages or 2′-O-methyl groups, may be able to cause silencing by actingin the nucleus. Examples of these types of modifications are describedin commonly assigned U.S. patent application Ser. Nos. 10/431,027 and10/613,077.

As described above, even when one selects at least two siRNAs at random,the effectiveness of the two may be greater than one would predict basedon the effectiveness of two individual siRNAs. This additive orsynergistic effect is particularly noticeable as one increases to atleast three siRNAs, and even more noticeable as one moves to at leastfour siRNAs. Surprisingly, the pooling of the non-functional andsemi-functional siRNAs, particularly more than five siRNAs, can lead toa silencing mixture that is as effective if not more effective than anyone particular functional siRNA.

Within the kits of the present invention, preferably each siRNA will bepresent in a concentration of between 0.001 and 200 μM, more preferablybetween 0.01 and 200 nM, and most preferably between 0.1 and 10 nM.

In addition to preferably comprising at least four or five siRNAs, thekits of the present invention will also preferably comprise a buffer tokeep the siRNA duplex stable. Persons skilled in the art are aware ofbuffers suitable for keeping siRNA stable. For example, the buffer maybe comprised of 100 mM KCl, 30 mM HEPES-pH 7.5, and 1 mM MgCl₂.Alternatively, kits might contain complementary strands that contain anyone of a number of chemical modifications (e.g., a 2′-O-ACE) thatprotect the agents from degradation by nucleases. In this instance, theuser may (or may not) remove the modifying protective group (e.g.,deprotect) before annealing the two complementary strands together.

By way of example, the kits may be organized such that pools of siRNAduplexes are provided on an array or microarray of wells or drops for aparticular gene set or for unrelated genes. The array may, for example,be in 96 wells, 384 wells or 1284 wells arrayed in a plastic plate or ona glass slide using techniques now known or that come to be known topersons skilled in the art. Within an array, preferably there will becontrols such as functional anti-lamin A/C, cyclophilin and two siRNAduplexes that are not specific to the gene of interest.

In order to ensure stability of the siRNA pools prior to usage, they maybe retained in lyophilized form at minus twenty degrees (−20° C.) untilthey are ready for use. Prior to usage, they should be resuspended;however, even once resuspended, for example, in the aforementionedbuffer, they should be kept at minus twenty degrees, (−20° C.) untilused. The aforementioned buffer, prior to use, may be stored atapproximately 4° C. or room temperature. Effective temperatures at whichto conduct transfections are well known to persons skilled in the artand include for example, room temperature.

The kits may be applied either in vivo or in vitro. Preferably, thesiRNA of the pools or kits is applied to a cell through transfection,employing standard transfection protocols. These methods are well knownto persons skilled in the art and include the use of lipid-basedcarriers, electroporation, cationic carriers, and microinjection.Further, one could apply the present invention by synthesizingequivalent DNA sequences (either as two separate, complementary strands,or as hairpin molecules) instead of siRNA sequences and introducing theminto cells through vectors. Once in the cells, the cloned DNA could betranscribed, thereby forcing the cells to generate the siRNA. Examplesof vectors suitable for use with the present application include but arenot limited to the standard transient expression vectors, adenoviruses,retroviruses, lentivirus-based vectors, as well as other traditionalexpression vectors. Any vector that has an adequate siRNA expression andprocession module may be used. Furthermore, certain chemicalmodifications to siRNAs, including but not limited to conjugations toother molecules, may be used to facilitate delivery. For certainapplications it may be preferable to deliver molecules withouttransfection by simply formulating in a physiological acceptablesolution.

This embodiment may be used in connection with any of the aforementionedembodiments. Accordingly, the sequences within any pool may be selectedby rational design.

Multigene Silencing

In addition to developing kits that contain multiple siRNA directedagainst a single gene, another embodiment includes the use of multiplesiRNA targeting multiple genes. Multiple genes may be targeted throughthe use of high- or hyper-functional siRNA. High- or hyper-functionalsiRNA that exhibit increased potency, require lower concentrations toinduce desired phenotypic (and thus therapeutic) effects. Thiscircumvents RISC saturation. It therefore reasons that if lowerconcentrations of a single siRNA are needed for knockout or knockdownexpression of one gene, then the remaining (uncomplexed) RISC will befree and available to interact with siRNA directed against two, three,four, or more, genes. Thus in this embodiment, the authors describe theuse of highly functional or hyper-functional siRNA to knock out threeseparate genes. More preferably, such reagents could be combined toknockout four distinct genes. Even more preferably, highly functional orhyperfunctional siRNA could be used to knock out five distinct genes.Most preferably, siRNA of this type could be used to knockout orknockdown the expression of six or more genes.

Hyperfunctional siRNA

The term hyperfunctional siRNA (hf-siRNA) describes a subset of thesiRNA population that induces RNAi in cells at low- or sub-nanomolarconcentrations for extended periods of time. These traits, heightenedpotency and extended longevity of the RNAi phenotype, are highlyattractive from a therapeutic standpoint. Agents having higher potencyrequire lesser amounts of the molecule to achieve the desiredphysiological response, thus reducing the probability of side effectsdue to “off-target” interference. In addition to the potentialtherapeutic benefits associated with hyperfunctional siRNA, hf-siRNA arealso desirable from an economic perspective. Hyperfunctional siRNA maycost less on a per-treatment basis, thus reducing overall expendituresto both the manufacturer and the consumer.

Identification of hyperfunctional siRNA involves multiple steps that aredesigned to examine an individual siRNA agent's concentration- and/orlongevity-profiles. In one non-limiting example, a population of siRNAdirected against a single gene are first analyzed using the previouslydescribed algorithm (Formula VIII). Individual siRNA are then introducedinto a test cell line and assessed for the ability to degrade the targetmRNA. It is important to note that when performing this step it is notnecessary to test all of the siRNA. Instead, it is sufficient to testonly those siRNA having the highest SMARTscores™ (i.e.,SMARTscore™>−10). Subsequently, the gene silencing data is plottedagainst the SMARTscores™ (see FIG. 9). siRNA that (1) induce a highdegree of gene silencing (i.e., they induce greater than 80% geneknockdown) and (2) have superior SMARTscores™ (i.e., a SMARTscore™of >−10, suggesting a desirable average internal stability profile) areselected for further investigations designed to better understand themolecule's potency and longevity. In one, non-limiting study dedicatedto understanding a molecule's potency, an siRNA is introduced into one(or more) cell types in increasingly diminishing concentrations (e.g.,3.0→0.3 nM). Subsequently, the level of gene silencing induced by eachconcentration is examined and siRNA that exhibit hyperfunctional potency(i.e., those that induce 80% silencing or greater at, e.g., picomolarconcentrations) are identified. In a second study, the longevityprofiles of siRNA having high (>−10) SMARTscores™ and greater than 80%silencing are examined. In one non-limiting example of how this isachieved, siRNA are introduced into a test cell line and the levels ofRNAi are measured over an extended period of time (e.g., 24-168 hrs).siRNAs that exhibit strong RNA interference patterns (i.e., >80%interference) for periods of time greater than, e.g., 120 hours, arethus identified. Studies similar to those described above can beperformed on any and all of the >10⁶ siRNA included in this document tofurther define the most functional molecule for any given gene.Molecules possessing one or both properties (extended longevity andheightened potency) are labeled “hyperfunctional siRNA,” and earmarkedas candidates for future therapeutic studies.

While the example(s) given above describe one means by whichhyperfunctional siRNA can be isolated, neither the assays themselves northe selection parameters used are rigid and can vary with each family ofsiRNA. Families of siRNA include siRNAs directed against a single gene,or directed against a related family of genes.

The highest quality siRNA achievable for any given gene may varyconsiderably. Thus, for example, in the case of one gene (gene X),rigorous studies such as those described above may enable theidentification of an siRNA that, at picomolar concentrations, induces99⁺% silencing for a period of 10 days. Yet identical studies of asecond gene (gene Y) may yield an siRNA that at high nanomolarconcentrations (e.g., 100 nM) induces only 75% silencing for a period of2 days. Both molecules represent the very optimum siRNA for theirrespective gene targets and therefore are designated “hyperfunctional.”Yet due to a variety of factors including but not limited to targetconcentration, siRNA stability, cell type, off-target interference, andothers, equivalent levels of potency and longevity are not achievable.Thus, for these reasons, the parameters described in the beforementioned assays can vary. While the initial screen selected siRNA thathad SMARTscore™ above −10 and a gene silencing capability of greaterthan 80%, selections that have stronger (or weaker) parameters can beimplemented. Similarly, in the subsequent studies designed to identifymolecules with high potency and longevity, the desired cutoff criteria(i.e., the lowest concentration that induces a desirable level ofinterference, or the longest period of time that interference can beobserved) can vary. The experimentation subsequent to application of therational criteria of this application is significantly reduced where oneis trying to obtain a suitable hyperfunctional siRNA for, for example,therapeutic use. When, for example, the additional experimentation ofthe type described herein is applied by one skilled in the art with thisdisclosure in hand, a hyperfunctional siRNA is readily identified.

The siRNA may be introduced into a cell by any method that is now knownor that comes to be known and that from reading this disclosure, personsskilled in the art would determine would be useful in connection withthe present invention in enabling siRNA to cross the cellular membrane.These methods include, but are not limited to, any manner oftransfection, such as, for example, transfection employing DEAE-Dextran,calcium phosphate, cationic lipids/liposomes, micelles, manipulation ofpressure, microinjection, electroporation, immunoporation, use ofvectors such as viruses, plasmids, cosmids, bacteriophages, cellfusions, and coupling of the polynucleotides to specific conjugates orligands such as antibodies, antigens, or receptors, passiveintroduction, adding moieties to the siRNA that facilitate its uptake,and the like.

Having described the invention with a degree of particularity, exampleswill now be provided. These examples are not intended to and should notbe construed to limit the scope of the claims in any way.

EXAMPLES

General Techniques and Nomenclatures

siRNA nomenclature. All siRNA duplexes are referred to by sense strand.The first nucleotide of the 5′-end of the sense strand is position 1,which corresponds to position 19 of the antisense strand for a 19-mer.In most cases, to compare results from different experiments, silencingwas determined by measuring specific transcript mRNA levels or enzymaticactivity associated with specific transcript levels, 24 hourspost-transfection, with siRNA concentrations held constant at 100 nM.For all experiments, unless otherwise specified, transfection efficiencywas ensured to be over 95%, and no detectable cellular toxicity wasobserved. The following system of nomenclature was used to compare andreport siRNA-silencing functionality: “F” followed by the degree ofminimal knockdown. For example, F50 signifies at least 50% knockdown,F80 means at least 80%, and so forth. For this study, all sub-F50 siRNAswere considered non-functional.

Cell culture and transfection. 96-well plates are coated with 50 μl of50 mg/ml poly-L-lysine (Sigma) for 1 hr, and then washed 3× withdistilled water before being dried for 20 min. HEK293 cells orHEK293Lucs or any other cell type of interest are released from theirsolid support by trypsinization, diluted to 3.5×10⁵ cells/ml, followedby the addition of 100 μL of cells/well. Plates are then incubatedovernight at 37° C., 5% CO₂. Transfection procedures can vary widelydepending on the cell type and transfection reagents. In onenon-limiting example, a transfection mixture consisting of 2 mL Opti-MEMI (Gibco-BRL), 80 μl Lipofectamine 2000 (Invitrogen), 15 μL SUPERNasinat 20 U/μl (Ambion), and 1.5 μl of reporter gene plasmid at 1 μg/μl isprepared in 5-ml polystyrene round bottom tubes. One hundred μl oftransfection reagent is then combined with 100 μl of siRNAs inpolystyrene deep-well titer plates (Beckman) and incubated for 20 to 30min at room temperature. Five hundred and fifty microliters of Opti-MEMis then added to each well to bring the final siRNA concentration to 100nM. Plates are then sealed with parafilm and mixed. Media is removedfrom HEK293 cells and replaced with 95 μl of transfection mixture. Cellsare incubated overnight at 37° C., 5% CO₂.

Quantification of gene knockdown. A variety of quantification procedurescan be used to measure the level of silencing induced by siRNA or siRNApools. In one non-limiting example: to measure mRNA levels 24 hrspost-transfection, QuantiGene branched-DNA (bDNA) kits (Bayer) (Wang, etal, Regulation of insulin preRNA splicing by glucose. Proc. Natl. Acad.Sci. USA 1997, 94:4360.) are used according to manufacturerinstructions. To measure luciferase activity, media is removed fromHEK293 cells 24 hrs post-transfection, and 50 μl of Steady-GLO reagent(Promega) is added. After 5 minutes, plates are analyzed on a platereader.

Example I Sequences Used to Develop the Algorithm

Anti-Firefly and anti-Cyclophilin siRNAs panels (FIG. 5 a, b) sortedaccording to using Formula VIII predicted values. All siRNAs scoringmore than 0 (formula VIII) and more then 20 (formula IX) are fullyfunctional. All ninety sequences for each gene (and DBI) appear below inTable III. TABLE III Cyclo 1 SEQ. ID 0032 GUUCCAAAAACAGUGGAUA Cyclo 2SEQ. ID 0033 UCCAAAAACAGUGGAUAAU Cyclo 3 SEQ. ID 0034CAAAAACAGUGGAUAAUUU Cyclo 4 SEQ. ID 0035 AAAACAGUGGAUAAUUUUG Cyclo 5SEQ. ID 0036 AACAGUGGAUAAUUUUGUG Cyclo 6 SEQ. ID 0037CAGUGGAUAAUUUUGUGGC Cyclo 7 SEQ. ID 0038 GUGGAUAAUUUUGUGGCCU Cyclo 8SEQ. ID 0039 GGAUAAUUUUGUGGCCUUA Cyclo 9 SEQ. ID 0040AUAAUUUUGUGGCCUUAGC Cyclo 10 SEQ. ID 0041 AAUUUUGUGGCCUUAGCUA Cyclo 11SEQ. ID 0042 UUUUGUGGCCUUAGCUACA Cyclo 12 SEQ. ID 0043UUGUGGCCUUAGCUACAGG Cyclo 13 SEQ. ID 0044 GUGGCCUUAGCUACAGGAG Cyclo 14SEQ. ID 0045 GGCCUUAGCUACAGGAGAG Cyclo 15 SEQ. ID 0046CCUUAGCUACAGGAGAGAA Cyclo 16 SEQ. ID 0047 UUAGCUACAGGAGAGAAAG Cyclo 17SEQ. ID 0048 AGCUACAGGAGAGAAAGGA Cyclo 18 SEQ. ID 0049CUACAGGAGAGAAAGGAUU Cyclo 19 SEQ. ID 0050 ACAGGAGAGAAAGGAUUUG Cyclo 20SEQ. ID 0051 AGGAGAGAAAGGAUUUGGC Cyclo 21 SEQ. ID 0052GAGAGAAAGGAUUUGGCUA Cyclo 22 SEQ. ID 0053 GAGAAAGGAUUUGGCUACA Cyclo 23SEQ. ID 0054 GAAAGGAUUUGGCUACAAA Cyclo 24 SEQ. ID 0055AAGGAUUUGGCUACAAAAA Cyclo 25 SEQ. ID 0056 GGAUUUGGCUACAAAAACA Cyclo 26SEQ. ID 0057 AUUUGGCUACAAAAACAGC Cyclo 27 SEQ. ID 0058UUGGCUACAAAAACAGCAA Cyclo 28 SEQ. ID 0059 GGCUACAAAAACAGCAAAU Cyclo 29SEQ. ID 0060 CUACAAAAACAGCAAAUUC Cyclo 30 SEQ. ID 0061ACAAAAACAGCAAAUUCCA Cyclo 31 SEQ. ID 0062 AAAAACAGCAAAUUCCAUC Cyclo 32SEQ. ID 0063 AAACAGCAAAUUCCAUCGU Cyclo 33 SEQ. ID 0064ACAGCAAAUUCCAUCGUGU Cyclo 34 SEQ. ID 0065 AGCAAAUUCCAUCGUGUAA Cyclo 35SEQ. ID 0066 CAAAUUCCAUCGUGUAAUC Cyclo 36 SEQ. ID 0067AAUUCCAUCGUGUAAUCAA Cyclo 37 SEQ. ID 0068 UUCCAUCGUGUAAUCAAGG Cyclo 38SEQ. ID 0069 CCAUCGUGUAAUCAAGGAC Cyclo 39 SEQ. ID 0070AUCGUGUAAUCAAGGACUU Cyclo 40 SEQ. ID 0071 CGUGUAAUCAAGGACUUCA Cyclo 41SEQ. ID 0072 UGUAAUCAAGGACUUCAUG Cyclo 42 SEQ. ID 0073UAAUCAAGGACUUCAUGAU Cyclo 43 SEQ. ID 0074 AUCAAGGACUUCAUGAUCC Cyclo 44SEQ. ID 0075 CAAGGACUUCAUGAUCCAG Cyclo 45 SEQ. ID 0076AGGACUUCAUGAUCCAGGG Cyclo 46 SEQ. ID 0077 GACUUCAUGAUCCAGGGCG Cyclo 47SEQ. ID 0078 CUUCAUGAUCCAGGGCGGA Cyclo 48 SEQ. ID 0079UCAUGAUCCAGGGCGGAGA Cyclo 49 SEQ. ID 0080 AUGAUCCAGGGCGGAGACU Cyclo 50SEQ. ID 0081 GAUCCAGGGCGGAGACUUC Cyclo 51 SEQ. ID 0082UCCAGGGCGGAGACUUCAC Cyclo 52 SEQ. ID 0083 CAGGGCGGAGACUUCACCA Cyclo 53SEQ. ID 0084 GGGCGGAGACUUCACCAGG Cyclo 54 SEQ. ID 0085GCGGAGACUUCACCAGGGG Cyclo 55 SEQ. ID 0086 GGAGACUUCACCAGGGGAG Cyclo 56SEQ. ID 0087 AGACUUCACCAGGGGAGAU Cyclo 57 SEQ. ID 0088ACUUCACCAGGGGAGAUGG Cyclo 58 SEQ. ID 0089 UUCACCAGGGGAGAUGGCA Cyclo 59SEQ. ID 0090 CACCAGGGGAGAUGGCACA Cyclo 60 SEQ. ID 0091CCAGGGGAGAUGGCACAGG Cyclo 61 SEQ. ID 0092 AGGGGAGAUGGCACAGGAG Cyclo 62SEQ. ID 0093 GGGAGAUGGCACAGGAGGA Cyclo 63 SEQ. ID 0094GAGAUGGCACAGGAGGAAA Cyclo 64 SEQ. ID 0095 GAUGGCACAGGAGGAAAGA Cyclo 65SEQ. ID 0431 UGGCACAGGAGGAAAGAGC Cyclo 66 SEQ. ID 0096GCACAGGAGGAAAGAGCAU Cyclo 67 SEQ. ID 0097 ACAGGAGGAAAGAGCAUCU Cyclo 68SEQ. ID 0098 AGGAGGAAAGAGCAUCUAC Cyclo 69 SEQ. ID 0099GAGGAAAGAGCAUCUACGG Cyclo 70 SEQ. ID 0100 GGAAAGAGCAUCUACGGUG Cyclo 71SEQ. ID 0101 AAAGAGCAUCUACGGUGAG Cyclo 72 SEQ. ID 0102AGAGCAUCUACGGUGAGCG Cyclo 73 SEQ. ID 0103 AGCAUCUACGGUGAGCGCU Cyclo 74SEQ. ID 0104 CAUCUACGGUGAGCGCUUC Cyclo 75 SEQ. ID 0105UCUACGGUGAGCGCUUCCC Cyclo 76 SEQ. ID 0106 UACGGUGAGCGCUUCCCCG Cyclo 77SEQ. ID 0107 CGGUGAGCGCUUCCCCGAU Cyclo 78 SEQ. ID 0108GUGAGCGCUUCCCCGAUGA Cyclo 79 SEQ. ID 0109 GAGCGCUUCCCCGAUGAGA Cyclo 80SEQ. ID 0110 GCGCUUCCCCGAUGAGAAC Cyclo 81 SEQ. ID 0111GCUUCCCCGAUGAGAACUU Cyclo 82 SEQ. ID 0112 UUCCCCGAUGAGAACUUCA Cyclo 83SEQ. ID 0113 CCCCGAUGAGAACUUCAAA Cyclo 84 SEQ. ID 0114CCGAUGAGAACUUCAAACU Cyclo 85 SEQ. ID 0115 GAUGAGAACUUCAAACUGA Cyclo 86SEQ. ID 0116 UGAGAACUUCAAACUGAAG Cyclo 87 SEQ. ID 0117AGAACUUCAAACUGAAGCA Cyclo 88 SEQ. ID 0118 AACUUCAAACUGAAGCACU Cyclo 89SEQ. ID 0119 CUUCAAACUGAAGCACUAC Cyclo 90 SEQ. ID 0120UCAAACUGAAGCACUACGG DB 1 SEQ. ID 0121 ACGGGCAAGGCCAAGUGGG DB 2 SEQ. ID0122 CGGGCAAGGCCAAGUGGGA DB 3 SEQ. ID 0123 GGGCAAGGCCAAGUGGGAU DB 4 SEQ.ID 0124 GGCAAGGCCAAGUGGGAUG DB 5 SEQ. ID 0125 GCAAGGCCAAGUGGGAUGC DB 6SEQ. ID 0126 CAAGGCCAAGUGGGAUGCC DB 7 SEQ. ID 0127 AAGGCCAAGUGGGAUGCGUDB 8 SEQ. ID 0128 AGGCCAAGUGGGAUGCCUG DB 9 SEQ. ID 0129GGCCAAGUGGGAUGCCUGG DB 10 SEQ. ID 0130 GCCAAGUGGGAUGCCUGGA DB 11 SEQ. ID0131 CCAAGUGGGAUGCCUGGAA DB 12 SEQ. ID 0132 CAAGUGGGAUGCCUGGAAU DB 13SEQ. ID 0133 AAGUGGGAUGCCUGGAAUG DB 14 SEQ. ID 0134 AGUGGGAUGCCUGGAAUGADB 15 SEQ. ID 0135 GUGGGAUGCCUGGAAUGAG DB 16 SEQ. ID 0136UGGGAUGCCUGGAAUGAGC DB 17 SEQ. ID 0137 GGGAUGCCUGGAAUGAGCU DB 18 SEQ. ID0138 GGAUGCCUGGAAUGAGCUG DB 19 SEQ. ID 0139 GAUGCCUGGAAUGAGCUGA DB 20SEQ. ID 0140 AUGCCUGGAAUGAGCUGAA DB 21 SEQ. ID 0141 UGCCUGGAAUGAGCUGAAADB 22 SEQ. ID 0142 GCCUGGAAUGAGCUGAAAG DB 23 SEQ. ID 0143CCUGGAAUGAGCUGAAAGG DB 24 SEQ. ID 0144 CUGGAAUGAGCUGAAAGGG DB 25 SEQ. ID0145 UGGAAUGAGCUGAAAGGGA DB 26 SEQ. ID 0146 GGAAUGAGCUGAAAGGGAC DB 27SEQ. ID 0147 GAAUGAGCUGAAAGGGACU DB 28 SEQ. ID 0148 AAUGAGCUGAAAGGGACUUDB 29 SEQ. ID 0149 AUGAGCUGAAAGGGACUUC DB 30 SEQ. ID 0150UGAGCUGAAAGGGACUUCC DB 31 SEQ. ID 0151 GAGCUGAAAGGGACUUCCA DB 32 SEQ. ID0152 AGCUGAAAGGGACUUCCAA DB 33 SEQ. ID 0153 GCUGAAAGGGACUUCCAAG DB 34SEQ. ID 0154 CUGAAAGGGACUUCCAAGG DB 35 SEQ. ID 0155 UGAAAGGGACUUCCAAGGADB 36 SEQ. ID 0156 GAAAGGGACUUCCAAGGAA DB 37 SEQ. ID 0157AAAGGGACUUCCAAGGAAG DB 38 SEQ. ID 0158 AAGGGACUUCCAAGGAAGA DB 39 SEQ. ID0159 AGGGACUUCCAAGGAAGAU DB 40 SEQ. ID 0160 GGGACUUCCAAGGAAGAUG DB 41SEQ. ID 0161 GGACUUCCAAGGAAGAUGC DB 42 SEQ. ID 0162 GACUUCCAAGGAAGAUGCCDB 43 SEQ. ID 0163 ACUUCCAAGGAAGAUGCCA DB 44 SEQ. ID 0164CUUCCAAGGAAGAUGCCAU DB 45 SEQ. ID 0165 UUCCAAGGAAGAUGCCAUG DB 46 SEQ. ID0166 UCCAAGGAAGAUGCCAUGA DB 47 SEQ. ID 0167 CCAAGGAAGAUGCCAUGAA DB 48SEQ. ID 0168 CAAGGAAGAUGCCAUGAAA DB 49 SEQ. ID 0169 AAGGAAGAUGCCAUGAAAGDB 50 SEQ. ID 0170 AGGAAGAUGCCAUGAAAGC DB 51 SEQ. ID 0171GGAAGAUGCCAUGAAAGCU DB 52 SEQ. ID 0172 GAAGAUGCCAUGAAAGCUU DB 53 SEQ. ID0173 AAGAUGCCAUGAAAGCUUA DB 54 SEQ. ID 0174 AGAUGCCAUGAAAGGUUAC DB 55SEQ. ID 0175 GAUGCCAUGAAAGCUUACA DB 56 SEQ. ID 0176 AUGCCAUGAAAGCUUACAUDB 57 SEQ. ID 0177 UGCCAUGAAAGCUUACAUC DB 58 SEQ. ID 0178GCCAUGAAAGCUUACAUCA DB 59 SEQ. ID 0179 CCAUGAAAGCUUACAUCAA DB 60 SEQ. ID0180 CAUGAAAGCUUACAUCAAC DB 61 SEQ. ID 0181 AUGAAAGCUUACAUCAACA DB 62SEQ. ID 0182 UGAAAGCUUACAUCAACAA DB 63 SEQ. ID 0183 GAAAGCUUACAUCAACAAADB 64 SEQ. ID 0184 AAAGCUUACAUCAACAAAG DB 65 SEQ. ID 0185AAGCUUACAUCAACAAAGU DB 66 SEQ. ID 0186 AGCUUACAUCAACAAAGUA DB 67 SEQ. ID0187 GCUUACAUCAACAAAGUAG DB 68 SEQ. ID 0188 CUUACAUCAACAAAGUAGA DB 69SEQ. ID 0189 UUACAUCAACAAAGUAGAA DB 70 SEQ. ID 0190 UACAUCAACAAAGUAGAAGDB 71 SEQ. ID 0191 ACAUCAACAAAGUAGAAGA DB 72 SEQ. ID 0192CAUCAACAAAGUAGAAGAG DB 73 SEQ. ID 0193 AUCAACAAAGUAGAAGAGC DB 74 SEQ. ID0194 UCAACAAAGUAGAAGAGCU DB 75 SEQ. ID 0195 CAACAAAGUAGAAGAGCUA DB 76SEQ. ID 0196 AACAAAGUAGAAGAGCUAA DB 77 SEQ. ID 0197 ACAAAGUAGAAGAGCUAAADB 78 SEQ. ID 0198 CAAAGUAGAAGAGCUAAAG DB 79 SEQ. ID 0199AAAGUAGAAGAGCUAAAGA DB 80 SEQ. ID 0200 AAGUAGAAGAGCUAAAGAA DB 81 SEQ. ID0201 AGUAGAAGAGCUAAAGAAA DB 82 SEQ. ID 0202 GUAGAAGAGCUAAAGAAAA DB 83SEQ. ID 0203 UAGAAGAGCUAAAGAAAAA DB 84 SEQ. ID 0204 AGAAGAGCUAAAGAAAAAADB 85 SEQ. ID 0205 GAAGAGCUAAAGAAAAAAU DB 86 SEQ. ID 0206AAGAGCUAAAGAAAAAAUA DB 87 SEQ. ID 0207 AGAGCUAAAGAAAAAAUAC DB 88 SEQ. ID0208 GAGCUAAAGAAAAAAUACG DB 89 SEQ. ID 0209 AGCUAAAGAAAAAAUACGG DB 90SEQ. ID 0210 GCUAAAGAAAAAAUACGGG Luc 1 SEQ. ID 0211 AUCCUCAUAAAGGCCAAGALuc 2 SEQ. ID 0212 AGAUCCUCAUAAAGGCCAA Luc 3 SEQ. ID 0213AGAGAUCCUCAUAAAGGCG Luc 4 SEQ. ID 0214 AGAGAGAUCCUCAUAAAGG Luc 5 SEQ. ID0215 UCAGAGAGAUCCUCAUAAA Luc 6 SEQ. ID 0216 AAUCAGAGAGAUCCUCAUA Luc 7SEQ. ID 0217 AAAAUCAGAGAGAUCCUCA Luc 8 SEQ. ID 0218 GAAAAAUCAGAGAGAUCCULuc 9 SEQ. ID 0219 AAGAAAAAUCAGAGAGAUC Luc 10 SEQ. ID 0220GCAAGAAAAAUCAGAGAGA Luc 11 SEQ. ID 0221 ACGCAAGAAAAAUCAGAGA Luc 12 SEQ.ID 0222 CGACGCAAGAAAAAUCAGA Luc 13 SEQ. ID 0223 CUCGACGCAAGAAAAAUCA Luc14 SEQ. ID 0224 AACUCGACGCAAGAAAAAU Luc 15 SEQ. ID 0225AAAACUCGACGCAAGAAAA Luc 16 SEQ. ID 0226 GGAAAACUCGACGCAAGAA Luc 17 SEQ.ID 0227 CCGGAAAACUCGACGCAAG Luc 18 SEQ. ID 0228 UACCGGAAAACUCGACGCA Luc19 SEQ. ID 0229 CUUACCGGAAAACUCGACG Luc 20 SEQ. ID 0230GUCUUACCGGAAAACUCGA Luc 21 SEQ. ID 0231 AGGUCUUACCGGAAAACUC Luc 22 SEQ.ID 0232 AAAGGUCUUACCGGAAAAC Luc 23 SEQ. ID 0233 CGAAAGGUCUUACCGGAAA Luc24 SEQ. ID 0234 ACCGAAAGGUCUUACCGGA Luc 25 SEQ. ID 0235GUACCGAAAGGUCUUACCG Luc 26 SEQ. ID 0236 AAGUACCGAAAGGUCUUAC Luc 27 SEQ.ID 0237 CGAAGUACCGAAAGGUCUU Luc 28 SEQ. ID 0238 GACGAAGUACCGAAAGGUC Luc29 SEQ. ID 0239 UGGACGAAGUACCGAAAGG Luc 30 SEQ. ID 0240UGUGGACGAAGUACCGAAA Luc 31 SEQ. ID 0241 UUUGUGGACGAAGUACCGA Luc 32 SEQ.ID 0242 UGUUUGUGGACGAAGUACC Luc 33 SEQ. ID 0243 UGUGUUUGUGGACGAAGUA Luc34 SEQ. ID 0244 GUUGUGUUUGUGGACGAAG Luc 35 SEQ. ID 0245GAGUUGUGUUUGUGGACGA Luc 36 SEQ. ID 0246 AGGAGUUGUGUUUGUGGAC Luc 37 SEQ.ID 0247 GGAGGAGUUGUGUUUGUGG Luc 38 SEQ. ID 0248 GCGGAGGAGUUGUGUUUGU Luc39 SEQ. ID 0249 GCGCGGAGGAGUUGUGUUU Luc 40 SEQ. ID 0250UUGCGCGGAGGAGUUGUGU Luc 41 SEQ. ID 0251 AGUUGCGCGGAGGAGUUGU Luc 42 SEQ.ID 0252 AAAGUUGCGCGGAGGAGUU Luc 43 SEQ. ID 0253 AAAAAGUUGCGCGGAGGAG Luc44 SEQ. ID 0254 CGAAAAAGUUGCGCGGAGG Luc 45 SEQ. ID 0255GGCGAAAAAGUUGCGCGGA Luc 46 SEQ. ID 0256 ACCGCGAAAAAGUUGCGCG Luc 47 SEQ.ID 0257 CAACCGCGAAAAAGUUGCG Luc 48 SEQ. ID 0258 AACAACCGCGAAAAAGUUG Luc49 SEQ. ID 0259 GUAACAACCGCGAAAAAGU Luc 50 SEQ. ID 0260AAGUAACAACCGCGAAAAA Luc 51 SEQ. ID 0261 UCAAGUAACAACCGCGAAA Luc 52 SEQ.ID 0262 AGUCAAGUAACAACCGCGA Luc 53 SEQ. ID 0263 CCAGUCAAGUAACAACCGC Luc54 SEQ. ID 0264 CGCCAGUCAAGUAACAACC Luc 55 SEQ. ID 0265GUCGCCAGUCAAGUAACAA Luc 56 SEQ. ID 0266 ACGUCGCCAGUCAAGUAAC Luc 57 SEQ.ID 0267 UUACGUCGGCAGUCAAGUA Luc 58 SEQ. ID 0268 GAUUACGUCGCCAGUCAAG Luc59 SEQ. ID 0269 UGGAUUACGUCGCCAGUCA Luc 60 SEQ. ID 0270CGUGGAUUACGUCGCCAGU Luc 61 SEQ. ID 0271 AUCGUGGAUUACGUCGCCA Luc 62 SEQ.ID 0272 AGAUCGUGGAUUACGUCGC Luc 63 SEQ. ID 0273 AGAGAUCGUGGAUUACGUC Luc64 SEQ. ID 0274 AAAGAGAUCGUGGAUUACG Luc 65 SEQ. ID 0275AAAAAGAGAUCGUGGAUUA Luc 66 SEQ. ID 0276 GGAAAAAGAGAUCGUGGAU Luc 67 SEQ.ID 0277 ACGGAAAAAGAGAUCGUGG Luc 68 SEQ. ID 0278 UGACGGAAAAAGAGAUCGU Luc69 SEQ. ID 0279 GAUGACGGAAAAAGAGAUC Luc 70 SEQ. ID 0280ACGAUGACGGAAAAAGAGA Luc 71 SEQ. ID 0281 AGACGAUGACGGAAAAAGA Luc 72 SEQ.ID 0282 AAAGACGAUGACGGAAAAA Luc 73 SEQ. ID 0283 GGAAAGACGAUGACGGAAA Luc74 SEQ. ID 0284 ACGGAAAGACGAUGACGGA Luc 75 SEQ. ID 0285GCAGGGAAAGACGAUGACG Luc 76 SEQ. ID 0286 GAGCACGGAAAGACGAUGA Luc 77 SEQ.ID 0287 UGGAGCACGGAAAGACGAU Luc 78 SEQ. ID 0288 UUUGGAGCACGGAAAGACG Luc79 SEQ. ID 0289 GUUUUGGAGCACGGAAAGA Luc 80 SEQ. ID 0290UUGUUUUGGAGCACGGAAA Luc 81 SEQ. ID 0291 UGUUGUUUUGGAGCACGGA Luc 82 SEQ.ID 0292 GUUGUUGUUUUGGAGCAGG Luc 83 SEQ. ID 0293 CCGUUGUUGUUUUGGAGCA Luc84 SEQ. ID 0294 CGCCGUUGUUGUUUUGGAG Luc 85 SEQ. ID 0295GCCGCCGUUGUUGUUUUGG Luc 86 SEQ. ID 0296 CCGGCGCCGUUGUUGUUUU Luc 87 SEQ.ID 0297 UCCCGCCGCCGUUGUUGUU Luc 88 SEQ. ID 0298 CUUCCCGCCGCCGUUGUUG Luc89 SEQ. ID 0299 AACUUCCCGCCGCCGUUGU Luc 90 SEQ. ID 0300UGAACUUCCCGCCGCCGUU

Example II Validation of the Algorithm Using DBI, Luciferase, PLK, EGFR,and SEAP

The algorithm (Formula VIII) identified siRNAs for five genes, humanDBI, firefly luciferase (fLuc), renilla luciferase (rLuc), human PLK,and human secreted alkaline phosphatase (SEAP). Four individual siRNAswere selected on the basis of their SMARTscores™ derived by analysis oftheir sequence using Formula VIII (all of the siRNAs would be selectedwith Formula IX as well) and analyzed for their ability to silence theirtargets' expression. In addition to the scoring, a BLAST search wasconducted for each siRNA. To minimize the potential for off-targetsilencing effects, only those target sequences with more than threemismatches against un-related sequences were selected. Semizarov, et al,Specificity of short interfering RNA determined through gene expressionsignatures. Proc. Natl. Acad. Sci. U.S.A. 2003, 100:6347. These duplexeswere analyzed individually and in pools of 4 and compared with severalsiRNAs that were randomly selected. The functionality was measured as apercentage of targeted gene knockdown as compared to controls. AllsiRNAs were transfected as described by the methods above at 100 nMconcentration into HEK293 using Lipofectamine 2000. The level of thetargeted gene expression was evaluated by B-DNA as described above andnormalized to the non-specific control. FIG. 10 shows that the siRNAsselected by the algorithm disclosed herein were significantly morepotent than randomly selected siRNAs. The algorithm increased thechances of identifying an F50 siRNA from 48% to 91%, and an F80 siRNAfrom 13% to 57%. In addition, pools of SMART siRNA silence the selectedtarget better than randomly selected pools (see FIG. 10F).

Example III Validation of the Algorithm Using Genes Involved inClathrin-Dependent Endocytosis

Components of clathrin-mediated endocytosis pathway are key tomodulating intracellular signaling and play important roles in disease.Chromosomal rearrangements that result in fusion transcripts between theMixed-Lineage Leukemia gene (MLL) and CALM (clathrin assembly lymphoidmyeloid leukemia gene) are believed to play a role in leukemogenesis.Similarly, disruptions in Rab7 and Rab9, as well as HIP1(Huntingtin-interacting protein), genes that are believed to be involvedin endocytosis, are potentially responsible for ailments resulting inlipid storage, and neuronal diseases, respectively. For these reasons,siRNA directed against clathrin and other genes involved in theclathrin-mediated endocytotic pathway are potentially important researchand therapeutic tools.

siRNAs directed against genes involved in the clathrin-mediatedendocytosis pathways were selected using Formula VIII. The targetedgenes were clathrin heavy chain (CHC, accession # NM_(—)004859),clathrin light chain A (CLCa, NM_(—)001833), clathrin light chain B(CLCb, NM_(—)001834), CALM (U45976), β2 subunit of AP-2 (β2,NM_(—)001282), Eps15 (NM_(—)001981), Eps15R (NM_(—)021235), dynamin II(DYNII, NM_(—)004945), Rab5a (BC001267), Rab5b (NM_(—)002868), Rab5c(AF141304), and EEA.1 (XM_(—)018197).

For each gene, four siRNAs duplexes with the highest scores wereselected and a BLAST search was conducted for each of them using theHuman EST database. In order to minimize the potential for off-targetsilencing effects, only those sequences with more than three mismatchesagainst un-related sequences were used. All duplexes were synthesized atDharmacon, Inc. as 21-mers with 3′-UU overhangs using a modified methodof 2′-ACE chemistry, Scaringe, Advanced 5′-silyl-2′-orthoester approachto RNA oligonucleotide synthesis, Methods Enzymol 2000, 317:3, and theantisense strand was chemically phosphorylated to insure maximizedactivity.

HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum, antibiotics and glutamine. siRNAduplexes were resuspended in 1× siRNA Universal buffer (Dharmacon, Inc.)to 20 μM prior to transfection. HeLa cells in 12-well plates weretransfected twice with 4 μl of 20 μM siRNA duplex in 3 μl Lipofectamine2000 reagent (Invitrogen, Carlsbad, Calif., USA) at 24-hour intervals.For the transfections in which 2 or 3 siRNA duplexes were included, theamount of each duplex was decreased, so that the total amount was thesame as in transfections with single siRNAs. Cells were plated intonormal culture medium 12 hours prior to experiments, and protein levelswere measured 2 or 4 days after the first transfection.

Equal amounts of lysates were resolved by electrophoresis, blotted, andstained with the antibody specific to targeted protein, as well asantibodies specific to unrelated proteins, PP1 phosphatase and Tsg101(not shown). The cells were lysed in Triton X-100/glycerolsolubilization buffer as described previously. Tebar, Bohlander, &Sorkin, Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein:Localization in Endocytic-coated Pits, Interactions with Clathrin, andthe Impact of Overexpression on Clathrin-mediated Traffic, Mol. Biol.Cell August 1999, 10:2687. Cell lysates were electrophoresed,transferred to nitrocellulose membranes, and Western blotting wasperformed with several antibodies followed by detection using enhancedchemiluminescence system (Pierce, Inc). Several x-ray films wereanalyzed to determine the linear range of the chemiluminescence signals,and the quantifications were performed using densitometry andAlphaImager v5.5 software (Alpha Innotech Corporation). In experimentswith Eps15R-targeted siRNAs, cell lysates were subjected toimmunoprecipitation with Ab860, and Eps15R was detected inimmunoprecipitates by Western blotting as described above.

The antibodies to assess the levels of each protein by Western blot wereobtained from the following sources: monoclonal antibody to clathrinheavy chain (TD.1) was obtained from American Type Culture Collection(Rockville, Md., USA); polyclonal antibody to dynamin II was obtainedfrom Affinity Bioreagents, Inc. (Golden, Colo., USA); monoclonalantibodies to EEA.1 and Rab5a were purchased from BD TransductionLaboratories (Los Angeles, Calif., USA); the monoclonal antibody toTsg101 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,Calif., USA); the monoclonal antibody to GFP was from ZYMED LaboratoriesInc. (South San Francisco, Calif., USA); the rabbit polyclonalantibodies Ab32 specific to α-adaptins and Ab20 to CALM were describedpreviously Sorkin, et al, Stoichiometric Interaction of the EpidermalGrowth Factor Receptor with the Clathrin-associated Protein ComplexAP-2, J. Biol. Chem. January 1995, 270:619, the polyclonal antibodies toclathrin light chains A and B were kindly provided by Dr. F. Brodsky(UCSF); monoclonal antibodies to PP1 (BD Transduction Laboratories) andα-Actinin (Chemicon) were kindly provided by Dr. M. Dell'Acqua(University of Colorado); Eps15 Ab577 and Eps15R Ab860 were kindlyprovided by Dr. P. P. Di Fiore (European Cancer Institute).

FIG. 11 demonstrates the in vivo functionality of 48 individual siRNAs,selected using Formula VIII (most of them will meet the criteriaincorporated by Formula IX as well) targeting 12 genes. Various celllines were transfected with siRNA duplexes (Dup1-4) or pools of siRNAduplexes (Pool), and the cells were lysed 3 days after transfection withthe exception of CALM (2 days) and β2 (4 days).

Note a β1-adaptin band (part of AP-1 Golgi adaptor complex) that runsslightly slower than β2 adaptin. CALM has two splice variants, 66 and 72kD. The full-length Eps15R (a doublet of ˜130 kD) and several truncatedspliced forms of ˜100 kD and ˜70 kD were detected in Eps15Rimmunoprecipitates (shown by arrows). The cells were lysed 3 days aftertransfection. Equal amounts of lysates were resolved by electrophoresisand blotted with the antibody specific to a targeted protein (GFPantibody for YFP fusion proteins) and the antibody specific to unrelatedproteins PP1 phosphatase or α-actinin, and TSG101. The amount of proteinin each specific band was normalized to the amount of non-specificproteins in each lane of the gel. Nearly all of them appear to befunctional, which establishes that Formula VIII and IX can be used topredict siRNAs' functionality in general in a genome wide manner.

To generate the fusion of yellow fluorescent protein (YFP) with Rab5b orRab5c (YFP-Rab5b or YFP-Rab5c), a DNA fragment encoding the full-lengthhuman Rab5b or Rab5c was obtained by PCR using Pfu polymerase(Stratagene) with a SacI restriction site introduced into the 5′ end anda KpnI site into the 3′ end and cloned into pEYFP-C1 vector (CLONTECH,Palo Alto, Calif., USA). GFP-CALM and YFP-Rab5a were describedpreviously Tebar, Bohlander, & Sorkin, Clathrin Assembly LymphoidMyeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits,Interactions with Clathrin, and the Impact of Overexpression onClathrin-mediated Traffic, Mol. Biol. Cell August 1999, 10:2687.

Example IV Validation of the Algorithm Using Eg5, GADPH, ATE1, MEK2,MEK1, QB, LaminA/C, c-myc, Human Cyclophilin, and Mouse Cyclophilin

A number of genes have been identified as playing potentially importantroles in disease etiology. Expression profiles of normal and diseasedkidneys has implicated Edg5 in immunoglobulin A neuropathy, a commonrenal glomerular disease. Myc1, MEK1/2 and other related kinases havebeen associated with one or more cancers, while lamins have beenimplicated in muscular dystrophy and other diseases. For these reasons,siRNA directed against the genes encoding these classes of moleculeswould be important research and therapeutic tools.

FIG. 12 illustrates four siRNAs targeting 10 different genes (Table Vfor sequence and accession number information) that were selectedaccording to the Formula VIII and assayed as individuals and pools inHEK293 cells. The level of siRNA induced silencing was measured usingthe B-DNA assay. These studies demonstrated that thirty-six out of theforty individual SMART-selected siRNA tested are functional (90%) andall 10 pools are fully functional.

Example V Validation of the Algorithm Using Bcl2

Bcl-2 is a ˜25 kD, 205-239 amino acid, anti-apoptotic protein thatcontains considerable homology with other members of the BCL familyincluding BCLX, MCL1, BAX, BAD, and BIK. The protein exists in at leasttwo forms (Bcl2a, which has a hydrophobic tail for membrane anchorage,and Bcl2b, which lacks the hydrophobic tail) and is predominantlylocalized to the mitochondrial membrane. While Bcl2 expression is widelydistributed, particular interest has focused on the expression of thismolecule in B and T cells. Bcl2 expression is down-regulated in normalgerminal center B cells yet in a high percentage of follicularlymphomas, Bcl2 expression has been observed to be elevated. Cytologicalstudies have identified a common translocation ((14;18)(q32;q32))amongst a high percentage (>70%) of these lymphomas. This genetic lesionplaces the Bcl2 gene in juxtaposition to immunoglobulin heavy chain gene(IgH) encoding sequences and is believed to enforce inappropriate levelsof gene expression, and resistance to programmed cell death in thefollicle center B cells. In other cases, hypomethylation of the Bcl2promoter leads to enhanced expression and again, inhibition ofapoptosis. In addition to cancer, dysregulated expression of Bcl-2 hasbeen correlated with multiple sclerosis and various neurologicaldiseases.

The correlation between Bcl-2 translocation and cancer makes this genean attractive target for RNAi. Identification of siRNA directed againstthe bcl2 transcript (or Bcl2-IgH fusions) would further ourunderstanding Bcl2 gene function and possibly provide a futuretherapeutic agent to battle diseases that result from altered expressionor function of this gene.

In Silico Identification of Functional siRNA.

To identify functional and hyperfunctional siRNA against the Bcl2 gene,the sequence for Bcl-2 was downloaded from the NCBI Unigene database andanalyzed using the Formula VIII algorithm. As a result of theseprocedures, both the sequence and SMARTscores™ of the Bcl2 siRNA wereobtained and ranked according to their functionality. Subsequently,these sequences were BLAST'ed (database) to insure that the selectedsequences were specific and contained minimal overlap with unrealatedgenes. The SMARTscores™ for the top 10 Bcl-2 siRNA are identified inFIG. 13.

In Vivo Testing of Bcl-2 SiRNA

Bcl-2 siRNAs having the top ten SMARTscores™ were selected and tested ina functional assay to determine silencing efficiency. To accomplishthis, each of the ten duplexes were synthesized using 2′-O-ACE chemistryand transfected at 100 nM concentrations into cells. Twenty-four hourslater assays were performed on cell extracts to assess the degree oftarget silencing. Controls used in these experiments included mocktransfected cells, and cells that were transfected with a non-specificsiRNA duplex.

The results of these experiments are presented below (and in FIG. 14)and show that all ten of the selected siRNA induce 80% or bettersilencing of the Bcl2 message at 100 nM concentrations. These dataverify that the algorithm successfully identified functional Bcl2 siRNAand provide a set of functional agents that can be used in experimentaland therapeutic environments. siRNA 1 GGGAGAUAGUGAUGAAGUA SEQ. ID NO.301 siRNA 2 GAAGUACAUCCAUUAUAAG SEQ. ID NO. 302 siRNA 3GUACGACAACCGGGAGAUA SEQ. ID NO. 303 siRNA 4 AGAUAGUGAUGAAGUACAU SEQ. IDNO. 304 siRNA 5 UGAAGACUCUGCUCAGUUU SEQ. ID NO. 305 siRNA 6GCAUGCGGCCUCUGUUUGA SEQ. ID NO. 306 siRNA 7 UGCGGCCUCUGUUUGAUUU SEQ. IDNO. 307 siRNA 8 GAGAUAGUGAUGAAGUACA SEQ. ID NO. 308 siRNA 9GGAGAUAGUGAUGAAGUAC SEQ. ID NO. 309 siRNA 10 GAAGACUCUGCUCAGUUUG SEQ. IDNO. 310

Bcl2 siRNA: Sense Strand, 5′→3′

Example VI Sequences Selected by the Algorithm

Sequences of the siRNAs selected using Formulas (Algorithms) VIII and IXwith their corresponding ranking, which have been evaluated for thesilencing activity in vivo in the present study (Formula VIII and IX,respectively) are shown in Table V. It should be noted that the “t”residues in Table V, and elsewhere, when referring to siRNA, should bereplaced by “u” residues. TABLE V Gene Accession Formula Formula NameNumber SEQ. ID NO. FTllSeqTence VIII IX CLTC NM_004859 SEQ. ID NO. 2400GAAAGAATCTGTAGAGAAA 76 94.2 CLTC NM_004859 SEQ. ID NO. 2401GCAATGAGCTGTTTGAAGA 65 39.9 CLTC NM_004859 SEQ. ID NO. 2402TGACAAAGGTGGATAAATT 57 38.2 CLTC NM_004859 SEQ. ID NO. 2403GGAAATGGATCTCTTTGAA 54 49.4 CLTA NM_001833 SEQ. ID NO. 2404GGAAAGTAATGGTCCAACA 22 55.5 CLTA NM_001833 SEQ. ID NO. 2405AGACAGTTATGCAGCTATT 4 22.9 CLTA NM_001833 SEQ. ID NO. 2406CCAATTCTCGGAAGCAAGA 1 17 CLTA NM_001833 SEQ. ID NO. 2407GAAAGTAATGGTCCAACAG −1 −13 CLTB NM_001834 SEQ. ID NO. 2408GCGCCAGAGTGAACAAGTA 17 57.5 CLTB NM_001834 SEQ. ID NO. 2409GAAGGTGGCCCAGCTATGT 15 −8.6 CLTB NM_001834 SEQ. ID NO. 0311GGAACCAGCGCCAGAGTGA 13 40.5 CLTB NM_001834 SEQ. ID NO. 0312GAGCGAGATTGCAGGCATA 20 61.7 CALM U45976 SEQ. ID NO. 0313GTTAGTATCTGATGACTTG 36 −34.6 CALM U45976 SEQ. ID NO. 0314GAAATGGAACCACTAAGAA 33 46.1 CALM U45976 SEQ. ID NO. 0315GGAAATGGAACCACTAAGA 30 61.2 CALM U45976 SEQ. ID NO. 0316CAACTACACTTTCCAATGC 28 6.8 EPS15 NM_001981 SEQ. ID NO. 0317CCACCAAGATTTCATGATA 48 25.2 EPS15 NM_001981 SEQ. ID NO. 0318GATCGGAACTCCAACAAGA 43 49.3 EPS15 NM_001981 SEQ. ID NO. 0319AAACGGAGCTACAGATTAT 39 11.5 EPS15 NM_001981 SEQ. ID NO. 0320CCACACAGCATTCTTGTAA 33 −23.6 EPS15R NM_021235 SEQ. ID NO. 0321GAAGTTACCTTGAGCAATC 48 33 EPS15R NM_021235 SEQ. ID NO. 0322GGACTTGGCCGATCCAGAA 27 33 EPS15R NM_021235 SEQ. ID NO. 0323GCACTTGGATCGAGATGAG 20 1.3 EPS15R NM_021235 SEQ. ID NO. 0324CAAAGACCAATTCGCGTTA 17 27.7 DNM2 NM_004945 SEQ. ID NO. 0325CCGAATCAATCGCATCTTC 6 −29.6 DNM2 NM_004945 SEQ. ID NO. 0326GACATGATCCTGCAGTTCA 5 −14 DNM2 NM_004945 SEQ. ID NO. 0327GAGCGAATCGTCACCACTT 5 24 DNM2 NM_004945 SEQ. ID NO. 0328CCTCCGAGCTGGCGTCTAC −4 −63.6 ARF6 AF93885 SEQ. ID NO. 0329TCACATGGTTAACCTCTAA 27 −21.1 ARF6 AF93885 SEQ. ID NO. 0330GATGAGGGACGCCATAATC 7 −38.4 ARF6 AF93885 SEQ. ID NO. 0331CCTCTAACTACAAATCTTA 4 16.9 ARF6 AF93885 SEQ. ID NO. 0332GGAAGGTGCTATCCAAAAT 4 11.5 RAB5A BC001267 SEQ. ID NO. 0333GCAAGCAAGTCCTAACATT 40 25.1 RAB5A BC001267 SEQ. ID NO. 0334GGAAGAGGAGTAGACCTTA 17 50.1 RAB5A BC001267 SEQ. ID NO. 0335AGGAATCAGTGTTGTAGTA 16 11.5 RAB5A BC001267 SEQ. ID NO. 0336GAAGAGGAGTAGACCTTAC 12 7 RAB5B NM_002868 SEQ. ID NO. 0337GAAAGTCAAGCCTGGTATT 14 18.1 RAB5B NM_002868 SEQ. ID NO. 0338AAAGTCAAGCCTGGTATTA 6 −17.8 RAB5B NM_002868 SEQ. ID NO. 0339GGTATGAACGTGAATGATC 3 −21.1 RAB5B NM_002868 SEQ. ID NO. 0340CAAGCCTGGTATTACGTTT −7 −37.5 RAB5C AF141304 SEQ. ID NO. 0341GGAACAAGATCTGTCAATT 38 51.9 RAB5C AF141304 SEQ. ID NO. 0342GCAATGAACGTGAACGAAA 29 43.7 RAB5C AF141304 SEQ. ID NO. 0343CAATGAACGTGAACGAAAT 18 43.3 RAB5C AF141304 SEQ. ID NO. 0344GGACAGGAGCGGTATCACA 6 18.2 EEA1 XM_018197 SEQ. ID NO. 0345AGACAGAGCTTGAGAATAA 67 64.1 EEA1 XM_018197 SEQ. ID NO. 0346GAGAAGATCTTTATGCAAA 60 48.7 EEA1 XM_018197 SEQ. ID NO. 0347GAAGAGAAATCAGCAGATA 58 45.7 EEA1 XM_018197 SEQ. ID NO. 0348GCAAGTAACTCAACTAACA 56 72.3 AP2B1 NM_001282 SEQ. ID NO. 0349GAGCTAATCTGCCACATTG 49 −12.4 AP2B1 NM_001282 SEQ. ID NO. 0350GCAGATGAGTTACTAGAAA 44 48.9 AP2B1 NM_001282 SEQ. ID NO. 0351CAACTTAATTGTCCAGAAA 41 28.2 AP2B1 NM_001282 SEQ. ID NO. 0352CAACACAGGATTCTGATAA 33 −5.8 PLK NM_005030 SEQ. ID NO. 0353AGATTGTGCCTAAGTCTCT −35 −3.4 PLK NM_005030 SEQ. ID NO. 0354ATGAAGATCTGGAGGTGAA 0 −4.3 PLK NM_005030 SEQ. ID NO. 0355TTTGAGACTTCTTGCCTAA −5 −27.7 PLK NM_005030 SEQ. ID NO. 0356AGATCACCCTCCTTAAATA 15 72.3 GAPDH NM_002046 SEQ. ID NO. 0357CAACGGATTTGGTCGTATT 27 −2.8 GAPDH NM_002046 SEQ. ID NO. 0358GAAATCCCATCACCATCTT 24 3.9 GAPDH NM_002046 SEQ. ID NO. 0359GACCTCAACTACATGGTTT 22 −22.9 GAPDH NM_002046 SEQ. ID NO. 0360TGGTTTACATGTTCCAATA 9 9.8 c-Myc SEQ. ID NO. 0361 GAAGAAATCGATGTTGTTT 31−11.7 c-Myc SEQ. ID NO. 0362 ACACAAACTTGAAGAGCTA 22 51.3 c-Myc SEQ. IDNO. 0363 GGAAGAAATCGATGTTGTT 18 26 c-Myc SEQ. ID NO. 0364GAAACGACGAGAACAGTTG 18 −8.9 MAP2K1 NM_002755 SEQ. ID NO. 0365GCACATGGATGGAGGTTCT 26 16 MAP2K1 NM_002755 SEQ. ID NO. 0366GCAGAGAGAGCAGATTTGA 16 0.4 MAP2K1 NM_002755 SEQ. ID NO. 0367GAGGTTCTCTGGATCAAGT 14 15.5 MAP2K1 NM_002755 SEQ. ID NO. 0368GAGCAGATTTGAAGCAACT 14 18.5 MAP2K2 NM_030662 SEQ. ID NO. 0369CAAAGACGATGACTTCGAA 37 26.4 MAP2K2 NM_030662 SEQ. ID NO. 0370GATCAGCATTTGCATGGAA 24 −0.7 MAP2K2 NM_030662 SEQ. ID NO. 0371TCCAGGAGTTTGTCAATAA 17 −4.5 MAP2K2 NM_030662 SEQ. ID NO. 0372GGAAGCTGATCCACCTTGA 16 59.2 KNSL1(EG5) NM_004523 SEQ. ID NO. 0373GCAGAAATCTAAGGATATA 53 35.8 KNSL1(EG5) NM_004523 SEQ. ID NO. 0374CAACAAGGATGAAGTCTAT 50 18.3 KNSL1(EGS) NM_004523 SEQ. ID NO. 0375CAGCAGAAATCTAAGGATA 41 32.7 KNSL1(EG5) NM_004523 SEQ. ID NO. 0376CTAGATGGCTTTCTCAGTA 39 3.9 CyclophilinA_ NM_021130 SEQ. ID NO. 0377AGACAAGGTCCCAAAGACA −16 58.1 CyclophilinA_ NM_021130 SEQ. ID NO. 0378GGAATGGCAAGACCAGCAA −6 36 CyclophilinA_ NM_021130 SEQ. ID NO. 0379AGAATTATTCCAGGGTTTA −3 16.1 CyclophilinA_ NM_021130 SEQ. ID NO. 0380GCAGACAAGGTCCCAAAGA 8 8.9 LAMIN A/C NM_170707 SEQ. ID NO. 0381AGAAGCAGCTTCAGGATGA 31 38.8 LAMIN A/C NM_170707 SEQ. ID NO. 0382GAGCTTGACTTCCAGAAGA 33 22.4 LAMIN A/C NM_170707 SEQ. ID NO. 0383CCACCGAAGTTCACCCTAA 21 27.5 LAMIN A/C NM_170707 SEQ. ID NO. 0384GAGAAGAGCTCCTCCATCA 55 30.1 CyclophilinB M60857 SEQ. ID NO. 0385GAAAGAGCATCTACGGTGA 41 83.9 CyclophilinB M60857 SEQ. ID NO. 0386GAAAGGATTTGGCTACAAA 53 59.1 CyclophilinB M60857 SEQ. ID NO. 0387ACAGCAAATTCCATCGTGT −20 28.8 CyclophilinB M60857 SEQ. ID NO. 0388GGAAAGACTGTTCCAAAAA 2 27 DBI1 NM_020548 SEQ. ID NO. 0389CAACACGCCTCATCCTCTA 27 −7.6 DBI2 NM_020548 SEQ. ID NO. 0390CATGAAAGCTTACATCAAC 25 −30.8 DBI3 NM_020548 SEQ. ID NO. 0391AAGATGCCATGAAAGCTTA 17 22 DBI4 NM_020548 SEQ. ID NO. 0392GCACATACCGCCTGAGTCT 15 3.9 rLUC1 SEQ. ID NO. 0393 GATCAAATCTGAAGAAGGA 5749.2 rLUC2 SEQ. ID NO. 0394 GCCAAGAAGTTTCCTAATA 50 13.7 rLUC3 SEQ. IDNO. 0395 CAGCATATCTTGAACCATT 41 −2.2 rLUC4 SEQ. ID NO. 0396GAACAAAGGAAACGGATGA 39 29.2 SeAP1 NM_031313 SEQ. ID NO. 0397CGGAAACGGTCCAGGCTAT 6 26.9 SeAP2 NM_031313 SEQ. ID NO. 0398GCTTCGAGCAGACATGATA 4 −11.2 SeAP3 NM_031313 SEQ. ID NO. 0399CCTACACGGTCCTCCTATA 4 4.9 SeAP4 NM_031313 SEQ. ID NO. 0400GCCAAGAACCTCATCATCT 1 −9.9 fLUC1 SEQ. ID NO. 0401 GATATGGGCTGAATACAAA 5440.4 fLUC2 SEQ. ID NO. 0402 GCACTCTGATTGACAAATA 47 54.7 fLUC3 SEQ. IDNO. 0403 TGAAGTCTCTGATTAAGTA 46 34.5 fLUC4 SEQ. ID NO. 0404TCAGAGAGATCCTCATAAA 40 11.4 mCyclo_1 NM_008907 SEQ. ID NO. 0405GCAAGAAGATCACCATTTC 52 46.4 mCyclo_2 NM_008907 SEQ. ID NO. 0406GAGAGAAATTTGAGGATGA 36 70.7 mCyclo_3 NM_008907 SEQ. ID NO. 0407GAAAGGATTTGGCTATAAG 35 −1.5 mCyclo_4 NM_008907 SEQ. ID NO. 0408GAAAGAAGGCATGAACATT 27 10.3 BCL2_1 NM_000633 SEQ. ID NO. 0409GGGAGATAGTGATGAAGTA 21 72 BCL2_2 NM_000633 SEQ. ID NO. 0410GAAGTACATCCATTATAAG 1 3.3 BCL2_3 NM_000633 SEQ. ID NO. 0411GTACGACAACCGGGAGATA 1 35.9 BCL2_4 NM_000633 SEQ. ID NO. 0412AGATAGTGATGAAGTACAT −12 22.1 BCL2_5 NM_000633 SEQ. ID NO. 0413TGAAGACTCTGCTCAGTTT 36 19.1 BCL2_6 NM_000633 SEQ. ID NO. 0414GCATGCGGCCTCTGTTTGA 5 −9.7 QB1 NM_003365.1 SEQ. ID NO. 0415GCACACAGCUUACUACAUC 52 −4.8 QB2 NM_003365.1 SEQ. ID NO. 0416GAAAUGCCCUGGUAUCUCA 49 22.1 QB3 NM_003365.1 SEQ. ID NO. 0417GAAGGAACGUGAUGUGAUC 34 22.9 QB4 NM_003365.1 SEQ. ID NO. 0418GCACUACUCCUGUGUGUGA 28 20.4 ATE1-1 NM_007041 SEQ. ID NO. 0419GAACCGAGCUGGAGAACUU 45 15.5 ATE1-2 NM_007041 SEQ. ID NO. 0420GAUAUACAGUGUGAUCUUA 40 12.2 ATE1-3 NM_007041 SEQ. ID NO. 0421GUACUACGAUCCUGAUUAU 37 32.9 ATE1-4 NM_007041 SEQ. ID NO. 0422GUGCCGACCUUUACAAUUU 35 18.2 EGFR-1 NM_005228 SEQ. ID NO. 0423GAAGGAAACTGAATTCAAA 68 79.4 EGFR-1 NM_005228 SEQ. ID NO. 0424GGAAATATGTACTACGAAA 49 49.5 EGFR-1 NM_005228 SEQ. ID NO. 0425CCACAAAGCAGTGAATTTA 41 7.6 EGFR-1 NM 005228 SEQ. ID NO. 0426GTAACAAGCTCACGCAGTT 40 25.9

Example VII Genome-Wide Application of Formula VIII or Formula X

The examples described above demonstrate that the algorithm(s) cansuccessfully identify functional siRNA and that these duplexes can beused to induce the desirable phenotype of transcriptional knockdown orknockout. Each gene or family of genes in each organism plays animportant role in maintaining physiological homeostasis and thealgorithm can be used to develop functional, highly functional, orhyperfunctional siRNA to each gene. In one example of how this isaccomplished, the entire online ncbi refseq, locuslink, and/or unigenedatabase for the human genome is first downloaded to local servers.Concommitantly, the most current version of the BLAST algorithm/programis also downloaded to enable analysis of all siRNA identified by thealgorithm. Prior to applying the algorithm, sequences are filtered toeliminate all non-coding sequences (e.g., 3′ and 5′ UTRs) and sequencesthat contain single nucleotide polymorphisms (SNPs). In addition, in oneversion of the siRNA selection process, only those sequences that areassociated with all isoforms (e.g., splice variants) of a given gene arereserved and considered for targeting. Subsequently, a list of allpotential siRNAs (including a 19 basepair “core” sequence with twobasepair 3′ overhangs) is generated for each gene sequence. This groupis then filtered to eliminate sequences that contain any one of a numberof undesirable traits including, but not limited to: 1) sequences thatcontain more than two GC basepairs in the last 5 nucleotides of the 3′end of the sense strand, and 2) sequences that contained internalrepeats that could potentially form hairpin structures. The output ofthese procedures are then submitted for scoring by the algorithm. Inthis example, the pre-filtered database was processed with Formula VIIIor Formula X and the top 5-100 siRNAs having scores of 75 (adjusted) orgreater were selected. If desired, the sequences of these siRNA can beBLAST'ed against the Unigene database containing all sequences in thegenome of choice (e.g., the human genome) to eliminate any duplexes thatshow undesirable degrees of homology to sequences other than theintended target. The sequences of the (roughly) top 100 sequences foreach gene are provided on the enclosed CDs in electronic form. In thisexample, the Formula X sequences were first generated using theprocedures described above and subsequently compared to Formula VIIIgenerated sequences. Formula VIII sequences that were also identified byFormula X were then removed (subtracted) from this database (Table XIII)to eliminate duplications.

With respect to the material on disk which is part of this disclosure,there are two tables provided in text format. Table XII, which islocated in a file entitled table-xii.txt, created 26 Apr. 2004, with afile size of 110,486 kb, provides a list of the 5-100 sequences for eachtarget, identified by Formula VIII as having the highest relativeSMARTscores™ for the target analyzed. Table XIII, which is located in afile entitled table-xiii.txt, created 26 Apr. 2004, with a file size of23,146 kb, provides a list of the 5-100 sequences for each targetidentified by Formula X. In addition, each table provides informationconcerning: the gene name, an NCBI accession number, an adjustedSMARTscore, and a sequence ID number. Any of the provided sequences canbe used for gene silencing either alone or in combination with othersequences. The information contained on the disks is part of this patentapplication and is incorporated into the specification by reference. Onemay use these tables in order to identify functional siRNAs for the geneprovided therein, by simply looking for the gene of interest and ansiRNA that is listed as functional. Preferably, one would select one ormore of the siRNAs that is most optimized for the target of interest andis denoted as a pool pick.

Table XII: siRNA Selected by Formula VIII

See data submitted herewith on a CD-ROM in accordance with PCTAdministrative Instructions Part 8. Table XII is included on the compactdisk labeled COPY 1—TABLES PART DISK 1/1, TABLES XII and XIII (providedin triplicate, which copies are identical), in a file entitledtable-xii.txt, date of creation 26 Apr. 2004, with a size of 110,486 kb.

Table XIII: siRNA Selected by Formula X

See data submitted herewith on a CD-ROM in accordance with PCTAdministrative Instructions Part 8. Table XIII is included on thecompact disk labeled COPY 1—TABLES PART DISK 1/1, TABLES XII and XIII(provided in triplicate, which copies are identical), in file entitledtable-xiii.txt, date of creation 26 Apr. 2004, with a size of 23,146 kb.

Many of the genes to which the described siRNA are directed playcritical roles in disease etiology. For this reason, the siRNAs listedin the accompanying compact disk may potentially act as therapeuticagents. A number of prophetic examples follow and should be understoodin view of the siRNA that are identified on the accompanying CD. Toisolate these siRNAs, the appropriate message sequence for each gene isanalyzed using one of the before mentioned formulas (preferably formulaVIII) to identify potential siRNA targets. Subsequently these targetsare BLAST'ed to eliminate homology with potential off-targets.

The list of potential disease targets is extensive. For instance,over-expression of Bcl10 has been implicated in the development of MALTlymphoma (mucosa associated lymphoid tissue lymphoma) and thus,functional, highly functional, or hyperfunctional siRNA directed againstthat gene (e.g., SEQ. ID NO. 0427: GGAAACCUCUCAUUGCUAA; SEQ. ID NO.0428: GAAAGAACCUUGCCGAUCA; SEQ. ID NO. 0429: GGAAAUACAUCAGAGCUUA, orSEQ. ID NO. 0430: GAAAGUAUGUGUCUUAAGU) may contribute to treatment ofthis disorder.

In another example, studies have shown that molecules that inhibitglutamine:fructose-6-phosphate aminotransferase (GFA) may act to limitthe symptoms suffered by Type II diabetics. Thus, functional, highlyfunctional, or hyperfunctional siRNA directed against GFA (also known asGFPT1: siRNA=SEQ. ID NO. 0433 UGAAACGGCUGCCUGAUUU; SEQ. ID NO. 0434GAAGUUACCUCUUACAUUU; SEQ. ID NO. 0435 GUACGAAACUGUAUGAUUA; SEQ. ID NO.0436 GGACGAGGCUAUCAUUAUG) may contribute to treatment of this disorder.

In another example, the von Hippel-Lindau (VHL) tumor suppressor hasbeen observed to be inactivated at a high frequency in sporadic clearcell renal cell carcinoma (RCC) and RCCs associated with VHL disease.The VHL tumor suppressor targets hypoxia-inducible factor-1 alpha (HIF-1alpha), a transcription factor that can induce vascular endothelialgrowth factor (VEGF) expression, for ubiquitination and degradation.Inactivation of VHL can lead to increased levels of HIF-1 alpha, andsubsequent VEGF over expression. Such over expression of VEGF has beenused to explain the increased (and possibly necessary) vascularityobserved in RCC. Thus, functional, highly functional, or hyperfunctionalsiRNAs directed against either HIF-1 alpha (SEQ. ID NO. 0437GAAGGAACCUGAUGCUUUA; SEQ. ID NO. 0438 GCAUAUAUCUAGAAGGUAU; SEQ. ID NO.0439 GAACAAAUACAUGGGAUUA; SEQ. ID NO. 0440 GGACACAGAUUUAGACUUG) or VEGF(SEQ. ID NO. 0441 GAACGUACUUGCAGAUGUG; SEQ. ID NO. 0442GAGAAAGCAUUUGUUUGUA; SEQ. ID NO. 0443 GGAGAAAGCAUUUGUUUGU; SEQ. ID NO.0444 CGAGGCAGCUUGAGUUAAA) may be useful in the treatment of renal cellcarcinoma.

In another example, gene expression of platelet derived growth factor Aand B (PDGF-A and PDGF-B) has been observed to be increased 22- and6-fold, respectively, in renal tissues taken from patients with diabeticnephropathy as compared with controls. These findings suggest that overexpression of PDGF A and B may play a role in the development of theprogressive fibrosis that characterizes human diabetic kidney disease.Thus, functional, highly functional, or hyperfunctional siRNAs directedagainst either PDGF A

(SEQ. ID NO. 0445: GGUAAGAUAUUGUGCUUUA;

SEQ. ID NO. 0446: CCGCAAAUAUGCAGAAUUA;

SEQ. ID NO. 0447: GGAUGUACAUGGCGUGUUA;

SEQ. ID NO. 0448: GGUGAAGUUUGUAUGUUUA) or

PDGF B

(SEQ. ID NO. 0449: CCGAGGAGCUUUAUGAGAU;

SEQ. ID NO. 0450: GCUCCGCGCUUUCCGAUUU;

SEQ. ID NO. 0451 GAGCAGGAAUGGUGAGAUG;

SEQ. ID NO. 0452: GAACUUGGGAUAAGAGUGU;

SEQ. ID NO. 0453 CCGAGGAGCUUUAUGAGAU;

SEQ. ID NO. 0454 UUUAUGAGAUGCUGAGUGA) may be useful in the treatment ofthis form of kidney disorder.

In another example, a strong correlation exists between theover-expression of glucose transporters (e.g., GLUT12) and cancer cells.It is predicted that cells undergoing uncontrolled cell growthup-regulate GLUT molecules so that they can cope with the heightenedenergy needs associated with increased rates of proliferation andmetastasis. Thus, siRNA-based therapies that target the molecules suchas GLUT1 (also known as SLC2A1: siRNA=

SEQ. ID NO.: 0455 GCAAUGAUGUCCAGAAGAA;

SEQ. ID NO.: 0456 GAAGAAUAUUCAGGACUUA;

SEQ. ID NO.: 0457 GAAGAGAGUCGGCAGAUGA;

SEQ. ID NO.: 0458 CCAAGAGUGUGCUAAAGAA)

GLUT12 (also known as SLCA12: siRNA=

SEQ. ID NO. 0459: GAGACACUCUGAAAUGAUA;

SEQ. ID NO. 0460: GAAAUGAUGUGGAUAAGAG;

SEQ. ID NO. 0461: GAUCAAAUCCUCCCUGAAA;

SEQ. ID NO. 0462: UGAAUGAGCUGAUGAUUGU) and other related transporters,may be of value in treating a multitude of malignancies.

The siRNA sequences listed above are presented in a 5′→3′ sense stranddirection. In addition, siRNA directed against the targets listed aboveas well as those directed against other targets and listed in theaccompanying compact disk may be useful as therapeutic agents.

Example VIII Evidence for the Benefits of Pooling

Evidence for the benefits of pooling have been demonstrated using thereporter gene, luciferase. Ninety siRNA duplexes were synthesized usingDharmacon proprietary ACE® chemistry against one of the standardreporter genes: firefly luciferase. The duplexes were designed to starttwo base pairs apart and to cover approximately 180 base pairs of theluciferase gene (see sequences in Table III). Subsequently, the siRNAduplexes were co-transfected with a luciferase expression reporterplasmid into HEK293 cells using standard transfection protocols andluciferase activity was assayed at 24 and 48 hours.

Transfection of individual siRNAs showed standard distribution ofinhibitory effect. Some duplexes were active, while others were not.FIG. 15 represents a typical screen of ninety siRNA duplexes (SEQ. IDNO. 0032-0120) positioned two base pairs apart. As the figure suggests,the functionality of the siRNA duplex is determined more by a particularsequence of the oligonucleotide than by the relative oligonucleotideposition within a gene or excessively sensitive part of the mRNA, whichis important for traditional anti-sense technology.

When two continuous oligonucleotides were pooled together, a significantincrease in gene silencing activity was observed. (See FIG. 16) Agradual increase in efficacy and the frequency of pools functionalitywas observed when the number of siRNAs increased to 3 and 4. (FIGS. 16,17). Further, the relative positioning of the oligonucleotides within apool did not determine whether a particular pool was functional (seeFIG. 18, in which 100% of pools of oligonucleotides distanced by 2, 10and 20 base pairs were functional).

However, relative positioning may nonetheless have an impact. Anincreased functionality may exist when the siRNA are positionedcontinuously head to toe (5′ end of one directly adjacent to the 3′ endof the others).

Additionally, siRNA pools that were tested performed at least as well asthe best oligonucleotide in the pool, under the experimental conditionswhose results are depicted in FIG. 19. Moreover, when previouslyidentified non-functional and marginally (semi) functional siRNAduplexes were pooled together in groups of five at a time, a significantfunctional cooperative action was observed. (See FIG. 20) In fact, poolsof semi-active oligonucleotides were 5 to 25 times more functional thanthe most potent oligonucleotide in the pool. Therefore, pooling severalsiRNA duplexes together does not interfere with the functionality of themost potent siRNAs within a pool, and pooling provides an unexpectedsignificant increase in overall functionality

Example IX Additional Evidence of the Benefits of Pooling

Experiments were performed on the following genes: β-galactosidase,Renilla luciferase, and Secreted alkaline phosphatase, whichdemonstrates the benefits of pooling. (see FIG. 21). Individual andpools of siRNA (described in Figure legend 21) were transfected intocells and tested for silencing efficiency. Approximately 50% ofindividual siRNAs designed to silence the above-specified genes werefunctional, while 100% of the pools that contain the same siRNA duplexeswere functional.

Example X Highly Functional siRNA

Pools of five siRNAs in which each two siRNAs overlap to 10-90% resultedin 98% functional entities (>80% silencing). Pools of siRNAs distributedthroughout the mRNA that were evenly spaced, covering an approximate20-2000 base pair range, were also functional. When the pools of siRNAwere positioned continuously head to tail relative to mRNA sequences andmimicked the natural products of Dicer cleaved long double stranded RNA,98% of the pools evidenced highly functional activity (>95% silencing).

Example XI Human Cyclophilin B

Table III above lists the siRNA sequences for the human cyclophilin Bprotein. A particularly functional siRNA may be selected by applyingthese sequences to any of Formula I to VII above.

Alternatively, one could pool 2, 3, 4, 5 or more of these sequences tocreate a kit for silencing a gene. Preferably, within the kit therewould be at least one sequence that has a relatively high predictedfunctionality when any of Formulas I-VII is applied.

Example XII Sample Pools of siRNAs and Their Application to HumanDisease

The genetic basis behind human disease is well documented and siRNA maybe used as both research or diagnostic tools and therapeutic agents,either individually or in pools. Genes involved in signal transduction,the immune response, apoptosis, DNA repair, cell cycle control, and avariety of other physiological functions have clinical relevance andtherapeutic agents that can modulate expression of these genes mayalleviate some or all of the associated symptoms. In some instances,these genes can be described as a member of a family or class of genesand siRNA (randomly, conventionally, or rationally designed) can bedirected against one or multiple members of the family to induce adesired result.

To identify rationally designed siRNA to each gene, the sequence wasanalyzed using Formula VIII or Formula X to identify rationally designedsiRNA. To confirm the activity of these sequences, the siRNA areintroduced into a cell type of choice (e.g., HeLa cells, HEK293 cells)and the levels of the appropriate message are analyzed using one ofseveral art proven techniques. siRNA having heightened levels of potencycan be identified by testing each of the before mentioned duplexes atincreasingly limiting concentrations. Similarly, siRNA having increasedlevels of longevity can be identified by introducing each duplex intocells and testing functionality at 24, 48, 72, 96, 120, 144, 168, and192 hours after transfection. Agents that induce >95% silencing atsub-nanomolar concentrations and/or induce functional levels ofsilencing for >96 hours are considered hyperfunctional.

Example XIII

The information presented in Tables XII and XIII provides the siRNAsequence (sense strand), the gene name, the NCBI accession number, theadjusted algorithm score, and the sequence ID number. All sequences havean adjusted score of 75 or above. For Table XIII, Formula X derivedsequences were compared with Formula VIII sequences. Sequences that werein common with both were eliminated from Table XIII. Pool picks aretypically identified as gene specific siRNA that have the hightestadjusted scores.

The following are non-limiting examples of families of proteins to whichsiRNA described in this document are targeted against:

Transporters, Pumps, and Channels

Transporters, pumps, and channels represent one class of genes that areattractive targets for siRNAs. One major class of transporter moleculesare the ATP-binding cassette (ABC) transporters. To date, nearly 50human ABC-transporter genes have been characterized and have been shownto be involved in a variety of physiological functions includingtransport of bile salts, nucleosides, chloride ions, cholesterol,toxins, and more. Predominant among this group are MDR1 (which encodesthe P-glycoprotein, NP_(—)000918), the MDR-related proteins (MRP1-7),and the breast cancer resistance protein (BCRP). In general, thesetransporters share a common structure, with each protein containing apair of ATP-binding domains (also known as nucleotide binding folds,NBF) and two sets of transmembrane (TM) domains, each of which typicallycontains six membrane-spanning α-helices. The genes encoding this classof transporter are organized as either full transporters (i.e.,containing two TM and two NBF domains) or as half transporters thatassemble as either homodimers or heterodimers to create functionaltransporters. As a whole, members of the family are widely dispersedthroughout the genome and show a high degree of amino acid sequenceidentify among eukaryotes.

ABC-transporters have been implicated in several human diseases. Forinstance, molecular efflux pumps of this type play a major role in thedevelopment of drug resistance exhibited by a variety of cancers andpathogenic microorganisms. In the case of human cancers, increasedexpression of the MDR1 gene and related pumps have been observed togenerate drug resistance to a broad collection of commonly usedchemotherapeutics including doxorubicin, daunorubicin, vinblastine,vincristine, colchicines. In addition to the contribution thesetransporters make to the development of multi-drug resistance, there arecurrently 13 human genetic diseases associated with defects in 14different transporters. The most common of these conditions includecystic fibrosis, Stargardt disease, age-related macular degeneration,adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome andprogressive familial intrahepatic cholestasis. For this reason, siRNAsdirected against members of this, and related, families are potentiallyvaluable research and therapeutic tools.

With respect to channels, analysis of Drosophila mutants has enabled theinitial molecular isolation and characterization of several distinctchannels including (but not limited to) potassium (K+) channels. Thislist includes shaker (Sh), which encodes a voltage activated K⁺ channel,slowpoke (Slo), a Ca²⁺ activated K⁺ channel, and ether-a-go-go (Eag).The Eag family is further divided into three subfamilies: Eag, Elk(eag-like K channels), and Erg (Eag related genes).

The Erg subfamily contains three separate family members (Erg1-3) thatare distantly related to the sh family of voltage activated K⁺ channels.Like sh, erg polypetides contain the classic six membrane spanningarchitecture of K⁺ channels (S1-S6) but differ in that each includes asegment associated with the C-terminal cytoplasmic region that ishomologous to cyclic nucleotide binding domains (cNBD). Like manyisolated ion channel mutants, erg mutants are temperature-sensitiveparalytics, a phenotype caused by spontaneous repetitive firing(hyperactivity) in neurons and enhanced transmitter release at theneuromuscular junction.

Initial studies on the tissue distribution of all three members of theerg subfamily show two general patterns of expression. Erg1 and erg3 arebroadly expressed throughout the nervous system and are observed in theheart, the superior mesenteric ganglia, the celiac ganglia, the retina,and the brain. In contrast, erg2 shows a much more restricted pattern ofexpression and is only observed in celiac ganglia and superiormesenteric ganglia. Similarly, the kinetic properties of the three ergpotassium channels are not homogeneous. Erg1 and erg2 channels arerelatively slow activating delayed rectifiers whereas the erg3 currentactivates rapidly and then exhibits a predominantly transient componentthat decays to a sustained plateau. The current properties of all threechannels are sensitive to methanesulfonanilides, suggesting a highdegree of conservation in the pore structure of all three proteins.

Recently, the erg family of K⁺ channels has been implicated in humandisease. Consistent with the observation that erg1 is expressed in theheart, single strand conformation polymorphism and DNA sequence analyseshave identified HERG (human erg1) mutations in six long-QT-syndrome(LQT) families, an inherited disorder that results in sudden death froma ventricular tachyarrythmia. Thus siRNA directed against this group ofmolecules (e.g., KCNH1-8) will be of extreme therapeutic value.

Another group of channels that are potential targets of siRNAs are theCLCA family that mediate a Ca²⁺-activated Cl⁻ conductance in a varietyof tissues. To date, two bovine (bCLC1; bCLCA2 (Lu-ECAM-1)), three mouse(mCLCA1; mCLCA2; mCLCA3) and four human (hCLCA1; hCLCA2; hCLCA3; hCLCA4)CLCA family members have been isolated and patch-clamp studies withtransfected human embryonic kidney (HEK-293) cells have shown thatbCLCA1, mCLCA1, and hCLCA1 mediate a Ca²⁺-activated Cl⁻ conductance thatcan be inhibited by the anion channel blocker DIDS and the reducingagent dithiothreitol (DTT).

The protein size, structure, and processing seem to be similar amongdifferent CLCA family members and has been studied in greatest detailfor Lu-ECAM-1. The Lu-ECAM-1 open reading frame encodes a precursorglycoprotein of 130 kDa that is processed to a 90-kDa amino-terminalcleavage product and a group of 30- to 40-kDa glycoproteins that areglycosylation variants of a single polypeptide derived from its carboxyterminus. Both subunits are associated with the outer cell surface, butonly the 90-kDa subunit is thought to be anchored to the cell membranevia four transmembrane domains.

Although the protein processing and function appear to be conservedamong CLCA homologs, significant differences exist in their tissueexpression patterns. For example, bovine Lu-ECAM-1 is expressedprimarily in vascular endothelia, bCLCA1 is exclusively detected in thetrachea, and hCLCA1 is selectively expressed in a subset of humanintestinal epithelial cells. Thus the emerging picture is that of amultigene family with members that are highly tissue specific, similarto the ClC family of voltage-gated Cl⁻ channels. The human channel,hCLCA2, is particular interesting from a medical and pharmacologicalstandpoint. CLCA2 is expressed on the luminal surface of lung vascularendothelia and serves as an adhesion molecule for lung metastatic cancercells, thus mediating vascular arrest and lung colonization. Expressionof this molecule in normal mammary epithelium is consistently lost inhuman breast cancer and in nearly all tumorigenic breast cancer celllines. Moreover, re-expression of hCLCA2 in human breast cancer cellsabrogates tumorigenicity in nude mice, implying that hCLCA2 acts as atumour suppressor in breast cancer. For these reasons, siRNA directedagainst CLCA family members and related channels may prove to bevaluable in research and therapeutic venues.

Transporters Involved in Synaptic Transmission

Synaptic transmission involves the release of a neurotransmitter intothe synaptic cleft, interaction of that transmitter with a postsynapticreceptor, and subsequent removal of the transmitter from the cleft. Inmost synapses the signal is terminated by a rapid reaccumulation of theneurotransmitter into presynaptic terminals. This process is catalyzedby specific neurotransmitter transporters that are often energized bythe electrochemical gradient of sodium across the plasma membrane of thepresynaptic cells.

Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in thecentral nervous system. The inhibitory action of GABA, mediated throughGABA_(A)/GABA_(B) receptors, and is regulated by GABA transporters(GATs), integral membrane proteins located perisynaptically on neuronsand glia. So far four different carriers (GAT1-GAT4) have been clonedand their cellular distribution has been partly worked out. Comparativesequence analysis has revealed that GABA transporters are related toseveral other proteins involved in neurotransmitter uptake includinggamma-aminobutyric acid transporters, monoamine transporters, amino acidtransporters, certain “orphan” transporters, and the recently discoveredbacterial transporters. Each of these proteins has a similar 12transmembrane helices topology and relies upon the Na+/Cl− gradient fortransport function. Transport rates are dependent on substrateconcentrations, with half-maximal effective concentrations for transportfrequently occurring in the submicromolar to low micromolar range. Inaddition, transporter function is bidirectional, and non-vesicularefflux of transmitter may contribute to ambient extracellulartransmitter levels.

Recent evidence suggests that GABA transporters, and neurotransmittertransporters in general, are not passive players in regulating neuronalsignaling; rather, transporter function can be altered by a variety ofinitiating factors and signal transduction cascades. In general, thisfunctional regulation occurs in two ways, either by changing the rate oftransmitter flux through the transporter or by changing the number offunctional transporters on the plasma membrane. A recurring theme intransporter regulation is the rapid redistribution of the transporterprotein between intracellular locations and the cell surface. Ingeneral, this functional modulation occurs in part through activation ofsecond messengers such as kinases, phosphatases, arachidonic acid, andpH. However, the mechanisms underlying transporter phosphorylation andtransporter redistribution have yet to be fully elucidated.

GABA transporters play a pathophysiological role in a number of humandiseases including temporal lobe epilepsy and are the targets ofpharmacological interventions. Studies in seizure sensitive animals showsome (but not all) of the GAT transporters have altered levels ofexpression at times prior to and post seizure, suggesting this class oftransporter may affect epileptogenesis, and that alterations followingseizure may be compensatory responses to modulate seizure activity. Forthese reasons, siRNAs directed against members of this family of genes(including but not limited to SLCG6A1-12) may prove to be valuableresearch and therapeutic tools.

Organic Ion Transporters

The human body is continuously exposed to a great variety ofxenobiotics, via food, drugs, occupation, and environment. Excretoryorgans such as kidney, liver, and intestine defend the body against thepotentially harmful effects of these compounds by transforming them intoless active metabolites that are subsequently secreted from the system.

Carrier-mediated transport of xenobiotics and their metabolites existfor the active secretion of organic anions and cations. Both systems arecharacterized by a high clearance capacity and tremendous diversity ofsubstances accepted, properties that result from the existance ofmultiple transporters with overlapping substrate specificities. Theclass of organic anion transporters plays a critical role in theelimination of a large number of drugs (e.g., antibiotics,chemotherapeutics, diuretics, nonsteroidal anti-inflammatory drugs,radiocontrast agents, cytostatics); drug metabolites (especiallyconjugation products with glutathione, glucuronide, glycine, sulfate,acetate); and toxicants and their metabolites (e.g., mycotoxins,herbicides, plasticizers, glutathione S-conjugates of polyhaloalkanes,polyhaloalkenes, hydroquinones, aminophenols), many of which arespecifically harmful to the kidney.

Over the past couple of years the number of identified aniontransporting molecules has grown tremendously. Uptake of organic anions(OA⁻) across the basolateral membrane is mediated by the classicsodium-dependent organic anion transport system, which includesα-ketoglutarate (α-KG²⁻)/OA⁻ exchange via the organic anion transporter(OAT1) and sodium-ketoglutarate cotransport via the Na⁺/dicarboxylatecotransporter (SDCT2). The organic anion transporting polypetide, Oatp1,and the kidney-specific OAT-K1 and OAT-K2 are seen as potentialmolecules that mediate facilitated OA⁻ efflux but could also be involvedin reabsorption via an exchange mechanism. Lastly the PEPT1 and PEPT2mediate luminal uptake of peptide drugs, whereas CNT1 and CNT2 areinvolved in reabsorption of nucleosides.

The organic anion-transporting polypeptide 1 (Oatp1) is a Na⁺- andATP-independent transporter originally cloned from rat liver. The tissuedistribution and transport properties of the Oatp1 gene product arecomplex. Oatp1 is localized to the basolateral membrane of hepatocytes,and is found on the apical membrane of S3 proximal tubules. Studies withtransiently transfected cells (e.g., HeLa cells) have indicated thatOatp1 mediates transport of a variety of molecules includingtaurocholate, estrone-3-sulfate, aldosterone, cortisol, and others. Theobserved uptake of taurocholate by Oatp1 expressed in X. laevis oocytesis accompanied by efflux of GSH, suggesting that transport by thismolecule may be glutathione dependent.

Computer modeling suggests that members of the Oatp family are highlyconserved, hydrophobic, and have 12 transmembrane domains. Decreases inexpression of Oatp family members have been associated with cholestaticliver diseases and human hepatoblastomas, making this family of proteinsof key interest to researchers and the medical community. For thesereasons, siRNAs directed against OAT family members (including but notlimited to SLC21A2, 3, 6, 8, 9, 11, 12, 14, 15, and relatedtransporters) are potentially useful as research and therapeutic tools.

Nucleoside Transporters

Nucleoside transporters play key roles in physiology and pharmacology.Uptake of exogenous nucleosides is a critical first step of nucleotidesynthesis in tissues such as bone marrow and intestinal epithelium andcertain parasitic organisms that lack de novo pathways for purinebiosynthesis. Nucleoside transporters also control the extracellularconcentration of adenosine in the vicinity of its cell surface receptorsand regulate processes such as neurotransmission and cardiovascularactivity. Adenosine itself is used clinically to treat cardiacarrhythmias, and nucleoside transport inhibitors such as dipyridamole,dilazep, and draflazine function as coronary vasodilators.

In mammals, plasma membrane transport of nucleosides is brought about bymembers of the concentrative, Na⁺-dependent (CNT) and equilibrative,Na⁺-independent (ENT) nucleoside transporter families. CNTs areexpressed in a tissue-specific fashion; ENTs are present in most,possibly all, cell types and are responsible for the movement ofhydrophilic nucleosides and nucleoside analogs down their concentrationgradients. In addition, structure/function studies of ENT family membershave predicted these molecules to contain eleven transmembrane helicalsegments with an amino terminus that is intracellular and a carboxylterminus that is extracellular. The proteins have a large glycosylatedloop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and7. Recent investigations have implicated the TM 3-6 region as playing acentral role in solute recognition. The medical importance of the ENTfamily of proteins is broad. In humans adenosine exerts a range ofcardioprotective effects and inhibitors of ENTs are seen as beingvaluable in alleviating a variety of cardio/cardiovascular ailments. Inaddition, responses to nucleoside analog drugs has been observed to varyconsiderably amongst, e.g., cancer patients. While some forms of drugresistance have been shown to be tied to the up-regulation ofABC-transporters (e.g., MDR1), resistance may also be the result ofreduced drug uptake (i.e., reduced ENT expression). Thus, a clearerunderstanding of ENT transporters may aid in optimizing drug treatmentsfor patients suffering a wide range of malignancies. For these reasons,siRNAs directed against this class of molecules (including SLC28A1-3,SLC29A1-4, and related molecules) may be useful as therapeutic andresearch tools.

Sulfate Transporters

All cells require inorganic sulfate for normal function. Sulfate is thefourth most abundant anion in human plasma and is the major source ofsulfur in many organisms. Sulfation of extracellular matrix proteins iscritical for maintaining normal cartilage metabolism and sulfate is animportant constituent of myelin membranes found in the brain

Because sulfate is a hydrophilic anion that cannot passively cross thelipid bilayer of cell membranes, all cells require a mechanism forsulfate influx and efflux to ensure an optimal supply. To date, avariety of sulfate transporters have been identified in tissues frommany origins. These include the renal sulfate transporters (NaSi-1 andSat-1), the ubiquitously expressed diastrophic dysplasia sulfatetransporter (DTDST), the intestinal sulfate transporter (DRA), and theerythrocyte anion exchanger (AE1). Most, if not all, of these moleculescontain the classic 12 transmembrane spanning domain architecturecommonly found amongst members of the anion transporter superfamily.

Recently three different sulfate transporters have been associated withspecific human genetic diseases. Family members SLC26A2, SLC26A3, andSLC26A4 have been recognized as the disease genes mutated in diastrophicdysplasia, congenital chloride diarrhea (CLD), and Pendred syndrome(PDS), respectively. DTDST is a particularly complex disorder. The geneencoding this molecule maps to chromosome 5q, and encodes two distincttranscripts due to alternative exon usage. In contrast to other sulfatetransporters (e.g., Sat-1) anion movement by the DTDST protein ismarkedly inhibited by either extracellular chloride or bicarbonate.Impaired function of the DTDST gene product leads to undersulfation ofproteoglycans and a complex family of recessively inheritedosteochondrodysplasias (achondrogenesis type 1B, atelosteogenesis typeII, and diastrophic dysplasia) with clinical features including but notlimited to, dwarfism, spinal deformation, and specific jointabnormalities. Interestingly, while epidemiological studies have shownthat the disease occurs in most populations, it is particularlyprevalent in Finland owing to an apparent founder effect. For thesereasons, siRNAs directed against this class of genes (including but notlimited to SLC26A1-9, and related molecules) may be potentially helpfulin both therapeutic and research venues.

Ion Exchangers

Intracellular pH regulatory mechanisms are critical for the maintenanceof countless cellular processes. For instance, in muscle cells,contractile processes and metabolic reactions are influenced by pH.During periods of increased energy demands and ischemia, muscle cellsproduce large amounts of lactic acid that, without quick and efficientdisposal, would lead to acidification of the sarcoplasm.

Several different transport mechanisms have evolved to maintain arelatively constant intracellular pH. The relative contribution of eachof these processes varies with cell type, the metabolic requirements ofthe cell, and the local environmental conditions. Intracellular pHregulatory processes that have been characterized functionally includebut are not limited to the Na⁺/H⁺ exchange, the Na(HCO₃)_(n)cotransport, and the Na⁺-dependent and -independent Cl⁻/base exchangers.As bicarbonate and CO₂ comprise the major pH buffer of biologicalfluids, sodium biocarbonate cotransporters (NBCs) are critical. Studieshave shown that these molecules exist in numerous tissues including thekidney, brain, liver, cornea, heart, and lung, suggesting that NBCs playan important role in mediating HCO₃ ⁻ transport in both epithelial aswell as nonepithelial cells.

Recent molecular cloning experiments have identified the existence offour NBC isoforms (NBC1, 2, 3 and 4) and two NBC-related proteins, AE4and NCBE (Anion Exchanger 4 and Na-dependent Chloride-BicarbonateExchanger). The secondary structure analyses and hydropathy profile ofthis family predict them to be intrinsic membrane proteins with 12putative transmembrane domains and several family members exhibitN-linked glycosylation sites, protein kinases A and C, casein kinase II,and ATP/GTP-binding consensus phosphorylation sites, as well aspotential sites for myristylation and amidation. AE4 is a relativelyrecent addition to this family of proteins and shows between 30-48%homology with the other family members. When expressed in COS-7 cellsand Xenopus oocytes AE4 exhibits sodium-independent and DIDS-insensitiveanion exchanger activity. Exchangers have been shown to be responsiblefor a variety of human diseases. For instance, mutations in three genesof the anion transporter family (SLC) are believed to cause knownhereditary diseases, including chondrodysplasia (SLC26A2, DTD), diarrhea(A3, down-regulated in adenoma/chloride-losing diarrhea protein:DRA/CLD), and goiter/deafness syndrome (A4, pendrin). Moreover,mutations in Na+/HCO3 co-transporters have also been associated withvarious human maladies. For these reasons, siRNAs directed against thesesorts of genes (e.g., SLC4A4-10, and related genes) may be useful fortherapeutic and research purposes.

Receptors Involved in Synaptic Transmission

In all vertebrates, fast inhibitory synaptic transmission is the resultof the interaction between the neurotransmitters glycine (Gly) andγ-aminobutyric acid (GABA) and their respective receptors. Thestrychnine-sensitive glycine receptor is especially important in that itacts in the mammalian spinal cord and brain stem and has awell-established role in the regulation of locomotor behavior.

Glycine receptors display significant sequence homology to several otherreceptors including the nicotinic acetylcholine receptor, theaminobutyric acid receptor type A (GABA_(A)R), and the serotoninreceptor type 3 (5-HT₃R) subunits. As members of the superfamily ofligand-gated ion channels, these polypeptides share common topologicalfeatures. The glycine receptor is composed of two types of glycosylatedintegral membrane proteins (α1-α4 and β) arranged in a pentamericsuprastructure. The alpha subunit encodes a large extracellular,N-terminal domain that carries the structural determinants essential foragonist and antagonist binding, followed by four transmembrane spanningregions (TM1-TM4), with TM2 playing the critical role of forming theinner wall of the chloride channel.

The density, location, and subunit composition of glycineneurotransmitter receptors changes over the course of development. Ithas been observed that the amount of GlyR gene translation (assessed bythe injection of developing rat cerebral cortex mRNA into Xenopusoocytes) decreases with age, whereas that of GABARs increases. Inaddition, the type and location of mRNAs coding for GlyR changes overthe course of development. For instance in a study of the expression ofalpha 1 and alpha 2 subunits in the rat, it was observed that (inembryonic periods E11-18) the mantle zone was scarce in the alpha 1mRNA, but the germinal zone (matrix layer) at E11-14 expressed higherlevels of the message. At postnatal day 0 (P0), the alpha 1 signalsbecame manifested throughout the gray matter of the spinal cord. Bycontrast, the spinal tissues at P0 exhibited the highest levels of alpha2 mRNA, which decreased with the postnatal development.

In both, man and mouse mutant lines, mutations of GlyR subunit genesresult in hereditary motor disorders characterized by exaggeratedstartle responses and increased muscle tone. Pathological alleles of theGlra1 gene are associated with the murine phenotypes oscillator(spd^(ot)) and spasmodic (spd). Similarly, a mutant allele of Glrb hasbeen found to underly the molecular pathology of the spastic mouse(spa). Resembling the situation in the mouse, a variety of GLRA1 mutantalleles have been shown to be associated with the human neurologicaldisorder hyperekplexia or startle disease. For these reasons, siRNAdirected against glycine receptors (GLRA1-3, GLRB, and relatedmolecules), glutamate receptors, GABA receptors, ATP receptors, andrelated neurotransmitter receptor molecules may be valuable therapeuticand research reagents.

Proteases

Kallikreins

One important class of proteases are the kallikreins, serineendopeptidases that split peptide substrates preferentially on theC-terminal side of internal arginyl and lysyl residues. Kallikreins aregenerally divided into two distinct groups, plasma kallikreins andtissue kallikreins. Tissue kallikreins represent a large group ofenzymes that have substantial similarities at both the gene and proteinlevel. The genes encoding this group are frequently found on a singlechromosome, are organized in clusters, and are expressed in a broadrange of tissues (e.g., pancreas, ovaries, breast). In contrast, theplasma form of the enzyme is encoded by a single gene (e.g., KLK3) thathas been localized to chromosome 4q34-35 in humans. The gene encodingplasma kallikrein is expressed solely in the liver, contains 15 exons,and encodes a glycoprotein that is translated as a preprotein calledprekallikrein.

Kallikreins are believed to play an important role in a host ofphysiological events. For instance, the immediate consequence of plasmaprekallikrein activation is the cleavage of high molecular weightkininogen (HK) and the subsequent liberation of bradykinin, a nine aminoacid vasoactive peptide that is an important mediator of inflammatoryresponses. Similarly, plasma kallikrein promotes single-chain urokinaseactivation and subsequent plasminogen activation, events that arecritical to blood coaggulation and wound healing.

Disruptions in the function of kallikreins have been implicated in avariety of pathological processes including imbalances in renal functionand inflammatory processes. For these reasons, siRNAs directed againstthis class of genes (e.g., KLK1-15) may prove valuable in both researchand therapeutic settings.

ADAM Proteins

The process of fertilization takes place in a series of discrete stepswhereby the sperm interacts with, i) the cumulus cells and thehyaluronic acid extracellular matrix (ECM) in which they are embedded,ii) the egg's own ECM, called the zona pellucida (ZP), and iii) the eggplasma membrane. During the course of these interactions, the “acrosomereaction,” the exocytosis of the acrosome vesicle on the head of thesperm, is induced, allowing the sperm to penetrate the ZP and gainaccess to the perivitelline space. This process exposes new portions ofthe sperm membrane, including the inner acrosomal membrane and theequatorial segment, regions of the sperm head that can participate ininitial gamete membrane binding.

The interactions of the gamete plasma membranes appear to involvemultiple ligands and receptors and are frequently compared toleukocyte-endothelial interactions. These interactions lead to a seriesof signal transduction events in the egg, known as collectively as eggactivation and include the initiation of oscillations in intracellularcalcium concentration, the exit from meiosis, the entry into the firstembryonic mitosis, and the formation of a block to polyspermy via therelease of ZP-modifying enzymes from the egg's cortical granules.Ultimately, sperm and egg not only adhere to each other but also go onto undergo membrane fusion, making one cell (the zygote) from two.

Studies on the process of sperm-egg interactions have identified anumber of proteins that are crucial for fertilization. One class ofproteins, called the ADAM family (A Disintegrin And Metalloprotease),has been found to be important in spermatogenesis and fertilization, aswell as various developmental systems including myogenesis andneurogenesis. Members of the family contain a disintegrin andmetalloprotease domain (and therefore have (potentially) both celladhesion and protease activities), as well as cysteine-rich regions,epidermal growth factor (EGF)-like domains, a transmembrane region, anda cytoplasmic tail. Currently, the ADAM gene family has 29 members andconstituents are widely distributed in many tissues including the brain,testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone,and muscle.

One of the best-studied members of the ADAM family is fertilin, aheterodimeric protein comprised of at least two subunits, fertilin alphaand fertilin beta. The fertilin beta gene (ADAM2) has been disruptedwith a targeting gene construct corresponding to the exon encoding thefertilin beta disintegrin domain. Sperm from males homozygous fordisruptions in this region exhibit defects in multiple facets of spermfunction including reduced levels of sperm transit from the uterus tothe oviduct, reduced sperm-ZP binding, and reduced sperm-egg binding,all of which contribute to male infertility.

Recently, four new ADAM family members (ADAM 24-27) have been isolated.The deduced amino acid sequences show that all four contain the completedomain organization common to ADAM family members and Northern Blotanalysis has shown all four to be specific to the testes. siRNAsdirected against this class of genes (e.g., ADAM2 and related proteins)may be useful as research tools and therapeutics directed towardfertility and birth control.

Aminopeptidases

Aminopeptidases are proteases that play critical roles in processes suchas protein maturation, protein digestion in its terminal stage,regulation of hormone levels, selective or homeostatic protein turnover,and plasmid stabilization. These enzymes generally have broad substratespecificity, occur in several forms and play a major role inphysiological homeostasis. For instance, the effects of bradykinin,angiotensin converting enzyme (ACE), and other vasoactive molecules aremuted by one of several peptidases that cleave the molecule at aninternal position and eliminate its ability to bind its cognate receptor(e.g., for bradykinin, the B2-receptor).

Among the enzymes that can cleave bradykinin is the membrane boundaminopeptidase P, also referred to as aminoacylproline aminopeptidase,proline aminopeptidase; X-Pro aminopeptidase (eukaryote) and XPNPEP2.Aminopeptidase P is an aminoacylproline aminopeptidase specific forNH₂-terminal Xaa-proline bonds. The enzyme i) is a mono-zinc-containingmolecule that lacks any of the typical metal binding motifs found inother zinc metalloproteases, ii) has an active-site configurationsimilar to that of other members of the MG peptidase family, and iii) ispresent in a variety of tissues including but not limited to the lung,kidney, brain, and intestine.

Aminopeptidases play an important role in a diverse set of humandiseases. Low plasma concentrations of aminopeptidase P are a potentialpredisposing factor for development of angio-oedema in patients treatedwith ACE inhibitors, and inhibitors of aminopeptidase P may act ascardioprotectors against other forms of illness including, but notlimited to myocardial infarction. For these reasons, siRNAs directedagainst this family of proteins (including but not limited to XPNPEP1and related proteins) may be useful as research and therapeutic tools.

Serine Proteases

One important class of proteases are the serine proteases. Serineproteases share a common catalytic triad of three amino acids in theiractive site (serine (nucleophile), aspartate (electrophile), andhistidine (base)) and can hydrolyze either esters or peptide bondsutilizing mechanisms of covalent catalysis and preferential binding ofthe transition state. Based on the position of their introns serineproteases have been classified into a minimum of four groups includingthose in which 1) the gene has no introns interrupting the exon codingfor the catalytic triad (e.g., the haptoglobin gene,); 2) each genecontains an intron just downstream from the codon for the histidineresidue at the active site, a second intron downstream from the exoncontaining the aspartic acid residue of the active site and a thirdintron just upstream from the exon containing the serine of the activesite (e.g., trypsinogen, chymotrypsinogen, kallikrein and proelastase);3) the genes contain seven introns interrupting the exons coding thecatalytic region (e.g., complement factor B gene); and 4) the genescontain two introns resulting in a large exon that contains both theactive site aspartatic acid and serine residues (e.g., factor X, factorIX and protein C genes).

Cytotoxic lymphocytes (e.g., CD8(+) cytotoxic T cells and natural killercells) form the major defense of higher organisms against virus-infectedand transformed cells. A key function of these cells is to detect andeliminate potentially harmful cells by inducing them to undergoapoptosis. This is achieved through two principal pathways, both ofwhich require direct but transient contact between the killer cell andits target. The first pathway involves ligation of TNF receptor-likemolecules such as Fas/CD95 to their cognate ligands, and results inmobilization of conventional, programmed cell-death pathways centered onactivation of pro-apoptotic caspases. The second mechanism consists of apathway whereby the toxic contents of a specialized class of secretoryvesicles are introduced into the target cell. Studies over the last twodecades have identified the toxic components as Granzymes, a family ofserine proteases that are expressed exclusively by cytotoxic Tlymphocytes and natural killer (NK) cells. These agents are stored inspecialized lytic granules and enter the target cell via endocytosis.Like caspases, cysteine proteases that play an important role inapoptosis, granzymes can cleave proteins after acidic residues,especially aspartic acid, and induce apoptosis in the recipient cell.

Granzymes have been grouped into three subfamilies according tosubstrate specificity. Members of the granzyme family that haveenzymatic activity similar to the serine protease chymotrypsin areencoded by a gene cluster termed the ‘chymase locus’. Similarly,granzymes with trypsin-like specificities are encoded by the ‘tryptaselocus’, and a third subfamily cleaves after unbranched hydrophobicresidues, especially methionine, and are encoded by the ‘Met-ase locus’.All granzymes are synthesized as zymogens and, after clipping of theleader peptide, obtain maximal enzymatic activity subsequent to theremoval of an amino-terminal dipeptide.

Granzymes have been found to be important in a number of importantbiological functions including defense against intracellular pathogens,graft versus host reactions, the susceptibility to transplantable andspontaneous malignancies, lymphoid homeostasis, and the tendency towardauto-immune diseases. For these reasons, siRNAs directed againstgranszymes (e.g., GZMA, GZMB, GZMH, GZHK, GZMM) and related serineproteases may be useful research and therapeutic reagents.

Kinases

Protein Kinases (PKs) have been implicated in a number of biologicalprocesses. Kinase molecules play a central role in modulating cellularphysiology and developmental decisions, and have been implicated in alarge list of human maladies including cancer, diabetes, and others.

During the course of the last three decades, over a hundred distinctprotein kinases have been identified, all with presumed specificcellular functions. A few of these enzymes have been isolated tosufficient purity to perform in vitro studies, but most remainintractable due to the low abundance of these molecules in the cell. Tocounter this technical difficulty, a number of protein kinases have beenisolated by molecular cloning strategies that utilize the conservedsequences of the catalytic domain to isolate closely related homologs.Alternatively, some kinases have been purified (and subsequentlystudied) based on their interactions with other molecules.

p58 is a member of the p34cdc2-related supergene family and contains alarge domain that is highly homologous to the cell division controlkinase, cdc2. This new cell division control-related protein kinase wasoriginally identified as a component of semipurifiedgalactosyltransferase; thus, it has been denotedgalactosyltransferase-associated protein kinase (GTA-kinase). GTA-kinasehas been found to be expressed in both adult and embryonic tissues andis known to phosphorylate a number of substrates, including histone H1,and casein. Interestingly enough, over expression of this molecule inCHO cells has shown that elevated levels of p58 result in a prolongedlate telophase and an early G1 phase, thus hinting of an important rolefor GTA-kinase in cell cycle regulation.

Cyclin Dependent Kinases

The cyclin-dependent kinases (Cdks) are a family of highly conservedserine/threonine kinases that mediate many of the cell cycle transitionsthat occur during duplication. Each of these Cdk catalytic subunitsassociates with a specific subset of regulatory subunits, termedcyclins, to produce a distinct Cdk.cyclin kinase complex that, ingeneral, functions to execute a unique cell cycle event.

Activation of the Cdk.cyclin kinases during cellular transitions iscontrolled by a variety of regulatory mechanisms. For the Cdc2.cyclin Bcomplex, inhibition of kinase activity during S phase and G₂ isaccomplished by phosphorylation of two Cdc2 residues, Thr¹⁴ and Tyr¹⁵,which are positioned within the ATP-binding cleft. Phosphorylation ofThr¹⁴ and/or Tyr¹⁵ suppresses the catalytic activity of the molecule bydisrupting the orientation of the ATP present within this cleft. Incontrast, the abrupt dephosphorylation of these residues by the Cdc25phosphatase results in the rapid activation of Cdc2.cyclin B kinaseactivity and subsequent downstream mitotic events. While the exactdetails of this pathway have yet to be elucidated, it has been proposedthat Thr¹⁴/Tyr¹⁵ phosphorylation functions to permit a cell to attain acritical concentration of inactive Cdk.cyclin complexes, which, uponactivation, induces a rapid and complete cell cycle transition.Furthermore, there is evidence in mammalian cells that Thr¹⁴/Tyr¹⁵phosphorylation also functions to delay Cdk activation after DNA damage.

The Schizosaccharomyces pombe wee1 gene product was the first kinaseidentified that is capable of phosphorylating Tyr¹⁵ in Cdc2. Homologs ofthe Wee1 kinase have been subsequently identified and biochemicallycharacterized from a wide range of species including human, mouse, frog,Saccharomyces cerevisiae, and Drosophila. In vertebrate systems, whereThr¹⁴ in Cdc2 is also phosphorylated, the Wee1 kinase was capable ofphosphorylating Cdc2 on Tyr¹⁵, but not Thr¹⁴, indicating that anotherkinase was responsible for Thr¹⁴ phosphorylation. This gene, Myt1kinase, was recently isolated from the membrane fractions of Xenopus eggextracts and has been shown to be capable of phosphorylating Thr¹⁴ and,to a lessor extent, Tyr¹⁵ in Cdc2. A human Myt1 homolog displayingsimilar properties has been isolated, as well as anon-membrane-associated molecule with Thr¹⁴ kinase activity.

In the past decade it has been shown that cancer can originate fromoverexpression of positive regulators, such as cyclins, or fromunderexpression of negative regulators (e.g., p16 (INK4a), p15 (INK4b),p21 (Cip1)). Inhibitors such as Myt1 are the focus of much cancerresearch because they are capable of controlling cell cycleproliferation, now considered the Holy Grail for cancer treatment. Forthese reasons, siRNA directed against kinases and kinase inhibitorsincluding but not limited to ABL1, ABL2, ACK1, ALK, AXL, BLK, BMX, BTK,C20orf64, CSF1R, SCK, DDR1, DDR2, DKFZp761P1010, EGFR, EPHA1, EPHA2,EPHA3, EPHA4, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4. EPHB6, ERBB2,ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3,FLT4, FRK, FYN, HCK, IGF1R, INSR, ITK, JAK1, JAK2, JAK3, KDR, KIAA1079,KIT, LCK, LTK, LYN, MATK, MERTK, MET, MST1R, MUSK, NTRK1, NTRK2, NTRK3,PDGFRA, PDGFRB, PTK2, PTK2B, PTK6, PTK7, PTK9, PTK9L, RET, ROR1, ROR2,ROS1, RYK, SRC, SYK, TEC, TEK, TIE, TNK1, TXK, TYK2, TYRO3, YES 1, andrelated proteins, may be useful for research and therapeutic purposes.

G Protein Coupled Receptors

One important class of genes to which siRNAs can be directed areG-protein coupled receptors (GPCRs). GPCRs constitute a superfamily ofseven transmembrane spanning proteins that respond to a diverse array ofsensory and chemical stimuli, such as light, odor, taste, pheromones,hormones and neurotransmitters. GPCRs play a central role in cellproliferation, differentiation, and have been implicated in the etiologyof disease.

The mechanism by which G protein-coupled receptors translateextracellular signals into cellular changes was initially envisioned asa simple linear model: activation of the receptor by agonist bindingleads to dissociation of the heterotrimeric GTP-binding G protein (Gs,Gi, or Gq) into its alpha and beta/gamma subunits, both of which canactivate or inhibit various downstream effector molecules. Morespecifically, activation of the GPCR induces a conformational change inthe Gα subunit, causing GDP to be released and GTP to be bound in itsplace. The Gα and Gβα subunits then dissociate from the receptor andinteract with a variety of effector molecules. For instance in the caseof the Gs family, the primary function is to stimulate the intracellularmessenger adenylate cyclase (AC), which catalyzes the conversion ofcytoplasmic ATP into the secondary messenger cyclic AMP (cAMP). Incontrast, the Gi family inhibits this pathway and the Gq familyactivates phospholipases C (PLC), which cleaves phosphatidylinositol4,5, bisphosphate (PIP2) to generate inositol-1,4,5-phosphate (IP3) anddiacylglycerol (DAG).

More recently, studies have shown that the functions of GPCRs are notlimited to their actions on G-proteins and that considerable cross-talkexists between this diverse group of receptor molecules and a secondclass of membrane bound proteins, the receptor tyrosine kinases (RTKs).A number of GPCRs such as endothelin-1, thrombin, bombesin, and dopaminereceptors can activate MAPKs, a downstream effector of the RTK/Raspathway. Interestingly, the interaction between these two families isnot unidirectional and RTKs can also modulate the activity of signalingpathways traditionally thought to be controlled exclusively by ligandsthat couple to GPCRs. For instance, EGF, which normally activates theMAPK cascade via the EGF receptor can stimulate adenylate cyclaseactivity by activating Gαs.

There are dozens of members of the G Protein-Coupled Receptor familythat have emerged as prominent drug targets in the last decade. Onenon-limiting list of potential GPCR-siRNA targets is as follows:

CMKLR1

CML1/CMKLR1 (Accession No. Q99788) is a member of the chemokine receptorfamily of GPCRs that may play a role in a number of diseases includingthose involved in inflammation and immunological responses (e.g.,asthma, arthritis). For this reason, siRNA directed against this proteinmay prove to be important therapeutic reagents.

Studies of juvenile-onset neuronal ceroid lipofuscinosis (JNCL, Battendisease), the most common form of childhood encephalopathy that ischaracterized by progressive neural degeneration, show that it isbrought on by mutations in a novel lysosomal membrane protein (CLN3). Inaddition to being implicated in JNCL, CLN3 (GPCR-like protein, AccessionNo. A57219) expression studies have shown that the CLN3 mRNA and proteinare highly over-expressed in a number of cancers (e.g., glioblastomas,neuroblastomas, as well as cancers of the prostate, ovaries, breast, andcolon) suggesting a possible contribution of this gene to tumor growth.For this reason, siRNA directed against this protein may prove to beimportant therapeutic reagents.

CLACR

The calcitonin receptor (CTR/CALCR, Accession No. NM_(—)001742) belongsto “family B” of GPCRs which typically recognized regulatory peptidessuch as parathyroid hormone, secretin, glucagons and vasoactiveintestinal polypeptide. Although the CT receptor typically binds tocalcitonin (CT), a 32 amino acid peptide hormone produced primarily bythe thyroid, association of the receptor with RAMP (Receptor ActivityModulating Protein) enables it to readily bind other members of thecalcitonin peptide family including amylin (AMY) and other CTgene-related peptides (e.g., αCGRP and βCGRP). While the primaryfunction of the calcitonin receptor pertains to regulating osteoclastmediated bone resorption and enhanced Ca⁺² excretion by the kidney,recent studies have shown that CT and CTRs may play an important role ina variety of processes as wide ranging as embryonic/fetal developmentand sperm function/physiology. In addition, studies have shown thatpatients with particular CTR genotypes may be at higher risk to losebone mass and that this GPCR may contribute to the formation of calciumoxalate urinary stones. For this reason, siRNA directed against CTR maybe useful as therapeutic reagents.

OXTR

The human oxytocin receptor (OTR, OXTR) is a 389 amino acid polypeptidethat exhibits the seven transmembrane domain structure and belongs tothe Class-I (rhodopsin-type) family of G-protein coupled receptors. OTRis expressed in a wide variety of tissues throughout development andmediates physiological changes through G(q) proteins and phospholipaseC-beta. Studies on the functions of oxytocin and the oxytocin receptorhave revealed a broad list of duties. OT and OTR play a role in a hostof sexual, maternal and social behaviors that include egg-laying, birth,milk-letdown, feeding, grooming, memory and learning. In addition, ithas been hypothesized that abnormalities in the functionality ofoxytocin-OTR receptor-ligand system can lead to a host of irregularitiesincluding compulsive behavior, eating disorders (such as anorexia),depression, and various forms of neurodegenerative diseases. For thesereasons, siRNA directed against this gene (NM_(—)000916) may play animportant role in combating OTR-associated illnesses.

EDG GPCRs

Lysophosphatidic acid and other lipid-based hormones/growth factorsinduce their effects by activating signaling pathways through theG-protein coupled receptors (GPCRs) and have been observed to playimportant roles in a number of human diseases including cancer, asthma,and vascular pathologies. For instance, during studies of immunoglobulinA nephropathy (IgAN), researchers have observed an enhanced expressionof EDG5 (NP_(—)004221) suggesting a contribution of this gene product inthe development of IgAN. For that reason, siRNA directed against Edg5(NM_(—)004230), Edg4 (NM_(—)004720), Edg7 (Nm_(—)012152) and relatedgenes may play an important role in combating human disease.

Genes Involved in Cholesterol Signaling and Biosynthesis

Studies on model genetic organisms such as Drosophila and C. eleganshave led to the identification of a plethora of genes that are essentialfor early development. Mutational analysis and ectopic expressionstudies have allowed many of these genes to be grouped into discreetsignal transduction pathways and have shown that these elements playcritical roles in pattern formation and cell differentiation. Disruptionof one or more of these genes during early stages of developmentfrequently leads to birth defects whereas as alteration of gene functionat later stages in life can result in tumorigenesis.

One critical set of interactions known to exist in both invertebratesand vertebrates is the Sonic Hedgehog-Patched-Gli pathway. Originallydocumented as a Drosophila segmentation mutant, several labs haverecently identified human and mouse orthologs of many of the pathwaysmembers and have successfully related disruptions in these genes toknown diseases. Pathway activation is initiated with the secretion ofSonic hedgehog. There are three closely related members of the Shhfamily (Sonic hedgehog, Desert, and Indian) with Shh being the mostwidely expressed form of the group. The Shh gene product is secreted asa small pro-signal molecule. To successfully initiate its developmentalrole, Shh is first cleaved, whereupon the N-terminal truncated fragmentis covalently modified with cholesterol. The addition of the sterolmoiety promotes the interaction between Shh and its cognate membranebound receptor, Patched (Ptch). There are at least two isoforms of thePatched gene, Ptch1 and Ptch2. Both isoforms contain a sterol-sensingdomain (SSD); a roughly 180 amino acid cluster that is found in at leastseven different classes of molecules including those involved incholesterol biosynthesis, vesicular traffic, signal transduction,cholesterol transport, and sterol homeostasis. In the absence of Shh,the Patched protein is a negative regulator of the pathway. In contrast,binding of Shh-cholesterol to the Patched receptor releases the negativeinhibition which that molecule enforces on a G-protein coupled receptorknown as Smoothened. Subsequent activation of Smoothened (directly orindirectly) leads to the triggering of a trio of transcription factorsthat belong to the Gli family. All three factors are relatively large,contain a characteristic C2-H2 zinc-finger pentamer, and recognize oneof two consensus sequences (SEQ. ID NO. 0463 GACCACCCA or SEQ. ID NO.0464 GAACCACCCA). In the absence of Shh, Gli proteins are cleaved by theproteosome and the C-terminally truncated fragment translocates to thenucleus and acts as a dominant transcription repressor. In the presenceof Shh-cholesterol, Gli repressor formation is inhibited and full-lengthGli functions as a transcriptional activator.

Shh and other members of the Shh-PTCH-Gli pathway are expressed in abroad range of tissues (e.g., the notochord, the floorplate of theneural tube, the brain, and the gut) at early stages in development. Notsurprisingly, mutations that lead to altered protein expression orfunction have been shown to induce developmental abnormalities. Defectsin the human Shh gene have been shown to cause holoprosencephaly, amidline defect that manifests itself as cleft lip or palate, CNSseptation, and a wide range of other phenotypes. Interestingly, defectsin cholesterol biosynthesis generate similar Shh-like disorders (e.g.,Smith-Lemli-Opitz syndrome) suggesting that cholesterol modification ofthe Shh gene product is crucial for pathway function. Both the Patchedand Smoothened genes have also been shown to be clinically relevant withSmoothened now being recognized as an oncogene that, like PTCH-1 andPTCH-2, is believed to be the causative agent of several forms of adulttumors. For these reasons, siRNA directed against Smoothened (SMO,NM_(—)005631), Patched (PTCH, nm_(—)000264), and additional genes thatparticipate in cholesterol signaling, biosynthesis, and degradation,have potentially useful research and therapeutic applications.

Targeted Pathways.

In addition to targeting siRNA against one or more members of a familyof proteins, siRNA can be directed against members of a pathway. Thus,for instance, siRNA can be directed against members of a signaltransduction pathway (e.g., the insulin pathway, including AKT1-3, CBL,CBLB, EIF4EBP1, FOXO1A, FOXO3A, FRAP1, GSK3A, GSK3B, IGF1, IGF1R,INPP5D, INSR, IRS1, MLLT7, PDPK1, PIK3CA, PIK3CB, PIK3R1, PIK3R2,PPP2R2B, PTEN, RPS6, RPS6KA1, RPX6KA3, SGK, TSC1, TSC2, AND XPO1), anapoptotic pathway (CASP3,6,7,8,9, DSH1/2, P110, P85, PDK1/2, CATENIN,HSP90, CDC37, P23, BAD, BCLXL, BCL2, SMAC, and others), pathways,involved in DNA damage, cell cycle, and other physiological (p53,MDM2,CHK1/2, BRCA1/2, ATM, ATR, P15INK4, P27, P21, SKP2, CDC25C/A, 14-3-3,PLK, RB, CDK4, GLUT4, Inos, Mtor, FKBP, PPAR, RXR, ER). Similarly, genesinvolved in immune system function including TNFR1, IL-IR, IRAK1/2,TRAF2, TRAF6, TRADD, FADD, IKKε, IKKγ, IKKβ, IKKα, IkBα, IkBβ, p50, p65,Rac, RhoA, Cdc42, ROCK, Pak1/2/3/4/5/6, cIAP, HDAC1/2, CBP, β-TrCP,Rip2/4, and others are also important targets for the siRNAs describedin this document and may be useful in treating immune system disorders.Genes involved in apoptosis, such as Dsh1/2,PTEN, P110 (pan), P85,PDK1/2, Akt1, Akt2, Akt (pan), p70^(S6K), GSK3β, PP2A (cat), β-catenin,HSP90, Cdc37/p50, P23, Bad, BclxL, Bcl2, Smac/Diablo, and Ask1 arepotentially useful in the treatment of diseases that involve defects inprogrammed cell death (e.g., cancer), while siRNA agents directedagainst p53, MDM2, Chk1/2, BRCA1/2, ATM, ATR, p15^(INK4), P27, P21,Skp2, Cdc25C/A, 14-3-3σ/ε, PLK, Rb, Cdk4, Glut4, iNOS, mTOR, FKBP,PPARγ, RXRα, ERα and related genes may play a critical role in combatingdiseases associated with disruptions in DNA repair, and cell cycleabnormalities.

Tables VI-Table X below provide examples of useful pools for inhibitingdifferent genes in the human insulin pathway and tyrosine kinasepathways, proteins involved in the cell cycle, the production of nuclearreceptors, and other genes. These particular pools are particularlyuseful in humans, but would be useful in any species that generates anappropriately homologous mRNA. Further, within each of the listed poolsany one sequence maybe used independently but preferably at least two ofthe listed sequences, more preferably at least three, and mostpreferably all of the listed sequences for a given gene is present.TABLE VI Gene SEQ. Name Acc # GI L.L. Duplex # Sequence ID NO. AKT1NM_005163 4885060 207 D-003000-05 GACAAGGACGGGCACATTA 465 AKT1 NM_0051634885060 207 D-003000-06 GGACAAGGACGGGCACATT 466 AKT1 NM_005163 4885060207 D-003000-07 GCTACTTCCTCCTCAAGAA 467 AKT1 NM_005163 4885060 207D-003000-08 GACCGCCTCTGCTTTGTCA 468 AKT2 AKT2 NM_001626 6715585 208D-003001-05 GTACTTCGATGATGAATTT 469 AKT2 NM_001626 6715585 208D-003001-06 GCAAAGAGGGCATCAGTGA 470 AKT2 NM_001626 6715585 208D-003001-07 GGGCTAAAGTGACCATGAA 471 AKT2 NM_001626 6715585 208D-003001-08 GCAGAATGCCAGCTGATGA 472 AKT3 AKT3 NM_005465 32307164 10000D-003002-05 GGAGTAAACTGGCAAGATG 473 AKT3 NM_005465 32307164 10000D-003002-06 GACATTAAATTTCCTCGAA 474 AKT3 NM_005465 32307164 10000D-003002-07 GACCAAAGCCAAACACATT 475 AKT3 NM_005465 32307164 10000D-003002-08 GAGGAGAGAATGAATTGTA 476 CBL CBL NM_005188 4885116 867D-003003-05 GGAGACACATTTCGGATTA 477 CBL NM_005188 4885116 867D-003003-06 GATCTGACCTGCAATGATT 478 CBL NM_005188 4885116 867D-003003-07 GACAATCCCTCACAATAAA 479 CBL NM_005188 4885116 867D-003003-08 CCAGAAAGCTTTGGTCATT 480 CBLB CBLB NM_170662 29366807 868D-003004-05 GACCATACCTCATAACAAG 481 CBLB NM_170662 29366807 868D-003004-06 TGAAAGACCTCCACCAATC 482 CBLB NM_170662 29366807 868D-003004-07 GATGAAGGCTCCAGGTGTT 483 CBLB NM_170662 29366807 868D-003004-08 TATCAGCATTTACGACTTA 484 EIF4EBP1 EIF4EBP1 NM_004095 200701791978 D-003005-05 GCAATAGCCCAGAAGATAA 485 EIF4EBP1 NM_004095 200701791978 D-003005-06 CGCAATAGCCCAGAAGATA 486 EIF4EBP1 NM_004095 200701791978 D-003005-07 GAGATGGACATTTAAAGCA 487 EIF4EBP1 NM_004095 200701791978 D-003005-08 CAATAGCCCAGAAGATAAG 488 FOXO1A FOXO1A NM_002015 92572212308 D-003006-05 CCAGGCATCTCATAACAAA 489 FOXO1A NM_002015 9257221 2308D-003006-06 CCAGATGCCTATACAAACA 490 FOXO1A NM_002015 9257221 2308D-003006-07 GGAGGTATGAGTCAGTATA 491 FOXO1A NM_002015 9257221 2308D-003006-08 GAGGTATGAGTCAGTATAA 492 FOXO3A FOXO3A NM_001455 4503738 2309D-003007-01 CAATAGCAACAAGTATACC 493 FOXO3A NM_001455 4503738 2309D-003007-02 TGAAGTCCAGGACGATGAT 494 FOXO3A NM_001455 4503738 2309D-003007-03 TGTCACACTATGGTAACCA 495 FOXO3A NM_001455 4503738 2309D-003007-04 TGTTCAATGGGAGCTTGGA 496 FRAP1 FRAP1 NM_004958 19924298 2475D-003008-05 GAGAAGAAATGGAAGAAAT 497 FRAP1 NM_004958 19924298 2475D-003008-06 CCAAAGTGCTGCAGTACTA 498 FRAP1 NM_004958 19924298 2475D-003008-07 GAGCATGCCGTCAATAATA 499 FRAP1 NM_004958 19924298 2475D-003008-08 GGTCTGAACTGAATGAAGA 500 GSK3A GSK3A NM_019884 11995473 2931D-003009-05 GGACAAAGGTGTTCAAATC 501 GSK3A NM_019884 11995473 2931D-003009-06 GAACCCAGCTGCCTAACAA 502 GSK3A NM_019884 11995473 2931D-003009-07 GCGCACAGCTTCTTTGATG 503 GSK3A NM_019884 11995473 2931D-003009-08 GCTCTAGCCTGCTGGAGTA 504 GSK3B GSK3B NM_002093 21361339 2932D-003010-05 GAAGAAAGATGAGGTCTAT 505 GSK3B NM_002093 21361339 2932D-003010-06 GGACCCAAATGTCAAACTA 506 GSK3B NM_002093 21361339 2932D-003010-07 GAAATGAACCCAAACTACA 507 GSK3B NM_002093 21361339 2932D-003010-08 GATGAGGTCTATCTTAATC 508 IGF1 IGF1 NM_000618 D-003011-05GGAAGTACATTTGAAGAAC 509 IGF1 NM_000618 D-003011-06 AGAAGGAAGTACATTTGAA510 IGF1 NM_000618 D-003011-07 CCTCAAGCCTGCCAAGTCA 511 IGF1 NM_000618D-003011-08 GGTGGATGCTCTTCAGTTC 512 IGF1R IGF1R NM_000875 11068002 3480D-003012-05 CAACGAAGCTTCTGTGATG 513 IGF1R NM_000875 11068002 3480D-003012-06 GGCCAGAAATGGAGAATAA 514 IGF1R NM_000875 11068002 3480D-003012-07 GAAGCACCCTTTAAGAATG 515 IGF1R NM_000875 11068002 3480D-003012-08 GCAGACACCTACAACATCA 516 INPP5D INPP5D NM_005541 5031798 3635D-003013-05 GGAATTGCGTTTACACTTA 517 INPP5D NM_005541 5031798 3635D-003013-06 GGAAACTGATCATTAAGAA 518 INPP5D NM_005541 5031798 3635D-003013-07 CGACAGGGATGAAGTACAA 519 INPP5D NM_005541 5031798 3635D-003013-08 AAACGCAGCTGCCCATCTA 520 INSR INSR NM_000208 4557883 3643D-003014-05 GGAAGACGTTTGAGGATTA 521 INSR NM_000208 4557883 3643D-003014-06 GAACAAGGCTCCCGAGAGT 522 INSR NM_000208 4557883 3643D-003014-07 GGAGAGACCTTGGAAATTG 523 INSR NM_000208 4557883 3643D-003014-08 GGACGGAACCCACCTATTT 524 IRS1 IRS1 NM_005544 5031804 3667D-003015-05 AAAGAGGTCTGGCAAGTGA 525 IRS1 NM_005544 5031804 3667D-003015-06 GAACCTGATTGGTATCTAC 526 IRS1 NM_005544 5031804 3667D-003015-07 CCACGGCGATCTAGTGCTT 527 IRS1 NM_005544 5031804 3667D-003015-08 GTCAGTCTGTCGTCCAGTA 528 MLLT7 MLLT7 NM_005938 5174578 4303D-003016-05 GGACTGGACTTCAACTTTG 529 MLLT7 NM_005938 5174578 4303D-003016-06 CCACGAAGCAGTTCAAATG 530 MLLT7 NM_005938 5174578 4303D-003016-07 GAGAAGCGACTGACACTTG 531 MLLT7 NM_005938 5174578 4303D-003016-08 GACCAGAGATCGCTAACCA 532 PDPK1 PDPK1 NM_002613 4505694 5170D-003017-05 CAAGAGACCTCGTGGAGAA 533 PDPK1 NM_002613 4505694 5170D-003017-06 GACCAGAGGCCAAGAATTT 534 PDPK1 NM_002613 4505694 5170D-003017-07 GGAAACGAGTATCTTATAT 535 PDPK1 NM_002613 4505694 5170D-003017-08 GAGAAGCGACATATCATAA 536 PIK3CA PIK3CA NM_006218 5453891 5290D-003018-05 GCTATCATCTGAACAATTA 537 PIK3CA NM_006218 5453891 5290D-003018-06 GGATAGAGGCCAAATAATA 538 PIK3CA NM_006218 5453891 5290D-003018-07 GGACAACTGTTTCATATAG 539 PIK3CA NM_006218 5453891 5290D-003018-08 GCCAGTACCTCATGGATTA 540 PIK3CB PIK3CB NM_006219 5453893 5291D-003019-05 CGACAAGACTGCCGAGAGA 541 PIK3CB NM_006219 5453893 5291D-003019-06 TCAAGTGTCTCCTAATATG 542 PIK3CB NM_006219 5453893 5291D-003019-07 GGATTCAGTTGGAGTGATT 543 PIK3CB NM_006219 5453893 5291D-003019-08 TTTCAAGTGTCTCCTAATA 544 PIK3R1 PIK3R1 NM_181504 324552515295 D-003020-05 GGAAATATGGCTTCTCTGA 545 PIK3R1 NM_181504 32455251 5295D-003020-06 GAAAGACGAGAGACCAATA 546 PIK3R1 NM_181504 32455251 5295D-003020-07 GTAAAGCATTGTGTCATAA 547 PIK3R1 NM_181504 32455251 5295D-003020-08 GGATCAAGTTGTCAAAGAA 548 PIK3R2 PIK3R2 NM_005027 4826907 5296D-003021-05 GGAAAGGCGGGAACAATAA 549 PIK3R2 NM_005027 4826907 5296D-003021-06 GATGAAGCGTACTGCAATT 550 PIK3R2 NM_005027 4826907 5296D-003021-07 GGACAGCGAATCTCACTAC 551 PIK3R2 NM_005027 4826907 5296D-003021-08 GCAAGATCCGAGACCAGTA 552 PPP2R2B PPP2R2B NM_004576 47589535521 D-003022-05 GAATGCAGCTTACTTTCTT 553 PPP2R2B NM_004576 4758953 5521D-003022-06 GACCGAAGCTGACATTATC 554 PPP2R2B NM_004576 4758953 5521D-003022-07 TCGATTACCTGAAGAGTTT 555 PPP2R2B NM_004576 4758953 5521D-003022-08 CCTGAAGAGTTTAGAAATA 556 PTEN PTEN NM_000314 4506248 5728D-003023-05 GTGAAGATCTTGACCAATG 557 PTEN NM_000314 4506248 5728D-003023-06 GATCAGCATACACAAATTA 558 PTEN NM_000314 4506248 5728D-003023-07 GGCGCTATGTGTATTATTA 559 PTEN NM_000314 4506248 5728D-003023-08 GTATAGAGCGTGCAGATAA 560 RPS6 RPS6 NM_001010 17158043 6194D-003024-05 GCCAGAAACTCATTGAAGT 561 RPS6 NM_001010 17158043 6194D-003024-06 GGATATTCCTGGACTGACT 562 RPS6 NM_001010 17158043 6194D-003024-07 CCAAGGAGAACTGGAGAAA 563 RPS6 NM_001010 17158043 6194D-003024-08 GCGTATGGCCACAGAAGTT 564 RPS6KA1 RPS6KA1 NM_002953 201495466195 D-003025-05 GATGACACCTTCTACTTTG 565 RPS6KA1 NM_002953 20149546 6195D-003025-06 GAGAATGGGCTCCTCATGA 566 RPS6KA1 NM_002953 20149546 6195D-003025-07 CAAGCGGGATCCTTCAGAA 567 RPS6KA1 NM_002953 20149546 6195D-003025-08 CCACCGGCCTGATGGAAGA 568 RPS6KA3 RPS6KA3 NM_004586 47590496197 D-003026-05 GAAGGGAAGTTGTATCTTA 569 RPS6KA3 NM_004586 4759049 6197D-003026-06 GAAAGTATGTGTATGTAGT 570 RPS6KA3 NM_004586 4759049 6197D-003026-07 GGACAGCATCCAAACATTA 571 RPS6KA3 NM_004586 4759049 6197D-003026-08 GGAGGTGAATTGCTGGATA 572 SGK SGK NM_005627 5032090 6446D-003027-01 TTAATGGTGGAGAGTTGTT 573 SGK NM_005627 5032090 6446D-003027-04 ATTAACTGGGATGATCTCA 574 SGK NM_005627 25168262 6446D-003027-05 GAAGAAAGCAATCCTGAAA 575 SGK NM_005627 25168262 6446D-003027-06 AAACACAGCTGAAATGTAC 576 TSC1 TSC1 NM_000368 24475626 7248D-003028-05 GAAGATGGCTATTCTGTGT 577 TSC1 NM_000368 24475626 7248D-003028-06 TATGAAGGCTCGAGAGTTA 578 TSC1 NM_000368 24475626 7248D-003028-07 CGACACGGCTGATAACTGA 579 TSC1 NM_000368 24475626 7248D-003028-08 CGGCTGATGTTGTTAAATA 580 TSC2 TSC2 NM_000548 10938006 7249D-003029-05 GCATTAATCTCTTACCATA 581 TSC2 NM_000548 10938006 7249D-003029-06 CCAATGTCCTCTTGTCTTT 582 TSC2 NM_000548 10938006 7249D-003029-07 GGAGACACATCACCTACTT 583 TSC2 NM_000548 10938006 7249D-003029-08 TCACCAGGCTCATCAAGAA 584 XPO1 XPO1 NM_003400 8051634 7514D-003030-05 GAAAGTCTCTGTCAAAATA 585 XPO1 NM_003400 8051634 7514D-003030-06 GCAATAGGCTCCATTAGTG 586 XPO1 NM_003400 8051634 7514D-003030-07 GGAACATGATCAACTTATA 587 XPO1 NM_003400 8051634 7514D-003030-08 GGATACAGATTCCATAAAT 588

TABLE VII Gene SEQ. Name Acc # GI L.L. Duplex # Sequence ID NO. ABL1ABL1 NM_007313 6382057 25 D-003100-05 GGAAATCAGTGACATAGTG 589 ABL1NM_007313 6382057 25 D-003100-06 GGTCCACACTGCAATGTTT 590 ABL1 NM_0073136382057 25 D-003100-07 GAAGGAAATCAGTGACATA 591 ABL1 NM_007313 6382057 25D-003100-08 TCACTGAGTTCATGACCTA 592 ABL2 ABL2 NM_007314 6382061 27D-003101-05 GAAATGGAGCGAACAGATA 593 ABL2 NM_007314 6382061 27D-003101-06 GAGCCAAATTTCCTATTAA 594 ABL2 NM_007314 6382061 27D-003101-07 GTAATAAGCCTACAGTCTA 595 ABL2 NM_007314 6382061 27D-003101-08 GGAGTGAAGTTCGCTCTAA 596 ACK1 ACK1 NM_005781 8922074 10188D-003102-05 AAACGCAAGTCGTGGATGA 597 ACK1 NM_005781 8922074 10188D-003102-06 GCAAGTCGTGGATGAGTAA 598 ACK1 NM_005781 8922074 10188D-003102-07 GAGCACTACCTCAGAATGA 599 ACK1 NM_005781 8922074 10188D-003102-08 TCAGCAGCACCCACTATTA 600 ALK ALK NM_004304 29029631 238D-003103-05 GACAAGATCCTGCAGAATA 601 ALK NM_004304 29029631 238D-003103-06 GGAAGAGTCTGGCAGTTGA 602 ALK NM_004304 29029631 238D-003103-07 GCACGTGGCTCGGGACATT 603 ALK NM_004304 29029631 238D-003103-08 GAACTGCAGTGAAGGAACA 604 AXL AXL NM_021913 21536465 558D-003104-05 GGTCAGAGCTGGAGGATTT 605 AXL NM_021913 21536465 558D-003104-06 GAAAGAAGGAGACCCGTTA 606 AXL NM_021913 21536465 558D-003104-07 CCAAGAAGATCTACAATGG 607 AXL NM_021913 21536465 558D-003104-08 GGAACTGCATGCTGAATGA 608 BLK BLK NM_001715 4502412 640D-003105-05 GAGGATGCCTGCTGGATTT 609 BLK NM_001715 4502412 640D-003105-06 ACATGAAGGTGGCCATTAA 610 BLK NM_001715 4502412 640D-003105-07 GGTCAGCGCCCAAGACAAG 611 BLK NM_001715 4502412 640D-003105-08 GAAACTCGGGTCTGGACAA 612 BMX BMX NM_001721 21359831 660D-003106-05 AAACAAACCTTTCCTACTA 613 BMX NM_001721 21359831 660D-003106-06 GAAGGAGCATTTATGGTTA 614 BMX NM_001721 21359831 660D-003106-07 GAGAAGAGATTACCTTGTT 615 BMX NM_001721 21359831 660D-003106-08 GTAAGGCTGTGAATGATAA 616 BTK BTK NM_000061 4557376 695D-003107-05 GAACAGGAATGGAAGCTTA 617 BTK NM_000061 4557376 695D-003107-06 GCTATGGGCTGCCAAATTT 618 BTK NM_000061 4557376 695D-003107-07 GAAAGCAACTTACCATGGT 619 BTK NM_000061 4557376 695D-003107-08 GGTAAACGATCAAGGAGTT 620 C20orf64 C20orf64 NM_033550 1992365511285 D-003108-05 CAACTTAGCCAAGACAATT 621 C20orf64 NM_033550 1992365511285 D-003108-06 GAAATTGAAGGCTCAGTGA 622 C20orf64 NM_033550 1992365511285 D-003108-07 TGGAACAGCTGAACATTGT 623 C20orf64 NM_033550 1992365511285 D-003108-08 GCTTCCAACTGCTTATATA 624 CSF1R CSF1R NM_005211 272626581436 D-003109-05 GGAGAGCTCTGACGTTTGA 625 CSF1R NM_005211 27262658 1436D-003109-06 CAACAACGCTACCTTCCAA 626 CSF1R NM_005211 27262658 1436D-003109-07 CCACGCAGCTGCCTTACAA 627 CSF1R NM_005211 27262658 1436D-003109-08 GGAACAACCTGCAGTTTGG 628 CSK CSK NM_004383 4758077 1445D-003110-05 CAGAATGTATTGCCAAGTA 629 CSK NM_004383 4758077 1445D-003110-06 GAACAAAGTCGCCGTCAAG 630 CSK NM_004383 4758077 1445D-003110-07 GCGAGTGCCTTATCCAAGA 631 CSK NM_004383 4758077 1445D-003110-08 GGAGAAGGGCTACAAGATG 632 DDR1 DDR1 NM_013994 7669484 780D-003111-05 GGAGATGGAGTTTGAGTTT 633 DDR1 NM_013994 7669484 780D-003111-06 CAGAGGCCCTGTCATCTTT 634 DDR1 NM_013994 7669484 780D-003111-07 GCTGGTAGCTGTCAAGATC 635 DDR1 NM_013994 7669484 780D-003111-08 TGAAAGAGGTGAAGATCAT 636 DDR2 DDR2 NM_006182 5453813 4921D-003112-05 GGTAAGAACTACACAATCA 637 DDR2 NM_006182 5453813 4921D-003112-06 GAACGAGAGTGCCACCAAT 638 DDR2 NM_006182 5453813 4921D-003112-07 ACACCAATCTGAAGTTTAT 639 DDR2 NM_006182 5453813 4921D-003112-08 CAACAAGAATGCCAGGAAT 640 DKFZp761 P1010 DKFZp761 NM_0184238922178 55359 D-003113-05 CCTAGAAGCTGCCATTAAA 641 P1010 DKFZp761NM_018423 8922178 55359 D-003113-06 GATTAGGCCTGGCTTATGA 642 P1010DKFZp761 NM_018423 8922178 55359 D-003113-07 CCCAGTAGCTGCACACATA 643P1010 DKFZp761 NM_018423 8922178 55359 D-003113-08 GGTGGTACCTGAACTGTAT644 P1010 EGFR EGFR NM_005228 4885198 1956 D-003114-05GAAGGAAACTGAATTCAAA 645 EGFR NM_005228 4885198 1956 D-003114-06GGAAATATGTACTACGAAA 646 EGFR NM_005228 4885198 1956 D-003114-07CCACAAAGCAGTGAATTTA 647 EGFR NM_005228 4885198 1956 D-003114-08GTAACAAGCTCACGCAGTT 648 EPHA1 EPHA1 NM_005232 4885208 2041 D-003115-05GACCAGAGCTTCACCATTC 649 EPHA1 NM_005232 4885208 2041 D-003115-06GCAAGACTGTGGCCATTAA 650 EPHA1 NM_005232 4885208 2041 D-003115-07GGGCGAACCTGACCTATGA 651 EPHA1 NM_005232 4885208 2041 D-003115-08GATTGTAGCCGTCATCTTT 652 EPHA2 EPHA2 NM_004431 4758277 1969 D-003116-05GGAGGGATCTGGCAACTTG 653 EPHA2 NM_004431 4758277 1969 D-003116-06GCAGCAAGGTGCACGAATT 654 EPHA2 NM_004431 4758277 1969 D-003116-07GGAGAAGGATGGCGAGTTC 655 EPHA2 NM_004431 4758277 1969 D-003116-08GAAGTTCACTACCGAGATC 656 EPHA3 EPHA3 NM_005233 21361240 2042 D-003117-05GATCGGACCTCCAGAAATA 657 EPHA3 NM_005233 21361240 2042 D-003117-06GAACTCAGCTCAGAAGATT 658 EPHA3 NM_005233 21361240 2042 D-003117-07GCAAGAGGCACAAATGTTA 659 EPHA3 NM_005233 21361240 2042 D-003117-08GAGCATCAGTTTACAAAGA 660 EPHA4 EPHA4 NM_004438 4758279 2043 D-003118-05GGTCTGGGATGAAGTATTT 661 EPHA4 NM_004438 4758279 2043 D-003118-06GAATGAAGTTACCTTATTG 662 EPHA4 NM_004438 4758279 2043 D-003118-07GAACTTGGGTGGATAGCAA 663 EPHA4 NM_004438 4758279 2043 D-003118-08GAGATTAAATTCACCTTGA 664 EPHA7 EPHA7 NM_004440 4758281 2045 D-003119-05GAAAAGAGATGTTGCAGTA 665 EPHA7 NM_004440 4758281 2045 D-003119-06CTAGATGCCTCCTGTATTA 666 EPHA7 NM_004440 4758281 2045 D-003119-07AGAAGAAGGTTATCGTTTA 667 EPHA7 NM_004440 4758281 2045 D-003119-08TAGCAAAGCTGACCAAGAA 668 EPHA8 EPHA8 NM_020526 18201903 2046 D-003120-05GAAGATGCACTATCAGAAT 669 EPHA8 NM_020526 18201903 2046 D-003120-06GAGAAGATGCACTATCAGA 670 EPHA8 NM_020526 18201903 2046 D-003120-07AACCTGATCTCCAGTGTGA 671 EPHA8 NM_020526 18201903 2046 D-003120-08TCTCAGACCTGGGCTATGT 672 EPHB1 EPHB1 NM_004441 21396502 2047 D-003121-05GCGATAAGCTCCAGCATTA 673 EPHB1 NM_004441 21396502 2047 D-003121-06GAAACGGGCTTATAGCAAA 674 EPHB1 NM_004441 21396502 2047 D-003121-07GGATGAAGATCTACATTGA 675 EPHB1 NM_004441 21396502 2047 D-003121-08GCACGTCTCTGTCAACATC 676 EPHB2 EPHB2 NM_017449 17975764 2048 D-003122-05ACTATGAGCTGCAGTACTA 677 EPHB2 NM_017449 17975764 2048 D-003122-06GTACAACGCCACAGCCATA 678 EPHB2 NM_017449 17975764 2048 D-003122-07GGAAAGCAATGACTGTTCT 679 EPHB2 NM_017449 17975764 2048 D-003122-08CGGACAAGCTGCAACACTA 680 EPHB3 EPHB3 NM_004443 17975767 2049 D-003123-05GGTGTGATCTCCAATGTGA 681 EPHB3 NM_004443 17975767 2049 D-003123-06GGGATGACCTCCTGTACAA 682 EPHB3 NM_004443 17975767 2049 D-003123-07CAGAAGACCTGCTCCGTAT 683 EPHB3 NM_004443 17975767 2049 D-003123-08GAGATGAAGTACTTTGAGA 684 EPHB4 EPHB4 NM_004444 17975769 2050 D-003124-05GGACAAACACGGACAGTAT 685 EPHB4 NM_004444 17975769 2050 D-003124-06GTACTAAGGTCTACATCGA 686 EPHB4 NM_004444 17975769 2050 D-003124-07GGAGAGAAGCAGAATATTC 687 EPHB4 NM_004444 17975769 2050 D-003124-08GCCAATAGCCACTCTAACA 688 EPHB6 EPHB6 NM_004445 4758291 2051 D-003125-05GGAAGTCGATCCTGCTTAT 689 EPHB6 NM_004445 4758291 2051 D-003125-06GGACCAAGGTGGACACAAT 690 EPHB6 NM_004445 4758291 2051 D-003125-07TGTGGGAAGTGATGAGTTA 691 EPHB6 NM_004445 4758291 2051 D-003125-08CGGGAGACCTTCACCCTTT 692 ERBB2 ERBB2 NM_004448 4758297 2064 D-003126-05GGACGAATTCTGCACAATG 693 ERBB2 NM_004448 4758297 2064 D-003126-06GACGAATTCTGCACAATGG 694 ERBB2 NM_004448 4758297 2064 D-003126-07CTACAACACAGACACGTTT 695 ERBB2 NM_004448 4758297 2064 D-003126-08AGACGAAGCATACGTGATG 696 ERBB3 ERBB3 NM_001982 4503596 2065 D-003127-05AAGAGGATGTCAACGGTTA 697 ERBB3 NM_001982 4503596 2065 D-003127-06GAAGACTGCCAGACATTGA 698 ERBB3 NM_001982 4503596 2065 D-003127-07GACAAACACTGGTGCTGAT 699 ERBB3 NM_001982 4503596 2065 D-003127-08GCAGTGGATTCGAGAAGTG 700 ERBB4 ERBB4 NM_005235 4885214 2066 D-003128-05GAGGAAAGATGCCAATTAA 701 ERBB4 NM_005235 4885214 2066 D-003128-06GCAGGAAACATCTATATTA 702 ERBB4 NM_005235 4885214 2066 D-003128-07GATCACAACTGCTGCTTAA 703 ERBB4 NM_005235 4885214 2066 D-003128-08CCTCAAAGATACCTAGTTA 704 FER FER NM_005246 4885230 2241 D-003129-05GGAGTGACCTGAAGAATTC 705 FER NM_005246 4885230 2241 D-003129-06TAAAGCAGATTCCCATTAA 706 FER NM_005246 4885230 2241 D-003129-07GGAAAGTACTGTCCAAATG 707 FER NM_005246 4885230 2241 D-003129-08GAACAACGGCTGCTAAAGA 708 FES FES NM_002005 13376997 2242 D-003130-05CGAGGATCCTGAAGCAGTA 709 FES NM_002005 13376997 2242 D-003130-06AGGAATACCTGGAGATTAG 710 FES NM_002005 13376997 2242 D-003130-07CAACAGGAGCTCCGGAATG 711 FES NM_002005 13376997 2242 D-003130-08GGTGTTGGGTGAGCAGATT 712 FGFR1 FGFR1 NM_000604 13186232 2260 D-003131-05TAAGAAATGTCTCCTTTGA 713 FGFR1 NM_000604 13186232 2260 D-003131-06GAAGACTGCTGGAGTTAAT 714 FGFR1 NM_000604 13186232 2260 D-003131-07GATGGTCCCTTGTATGTCA 715 FGFR1 NM_000604 13186232 2260 D-003131-08CTTAAGAAATGTCTCCTTT 716 FGFR2 FGFR2 NM_000141 13186239 2263 D-003132-05CCAAATCTCTCAACCAGAA 717 FGFR2 NM_000141 13186239 2263 D-003132-06GAACAGTATTCACCTAGTT 718 FGFR2 NM_000141 13186239 2263 D-003132-07GGCCAACACTGTCAAGTTT 719 FGFR2 NM_000141 13186239 2263 D-003132-08GTGAAGATGTTGAAAGATG 720 FGFR3 FGFR3 NM_000142 13112046 2261 D-003133-05TGTCGGACCTGGTGTCTGA 721 FGFR3 NM_000142 13112046 2261 D-003133-06GCATCAAGCTGCGGCATCA 722 FGFR3 NM_000142 13112046 2261 D-003133-07GGACGGCACACCCTACGTT 723 FGFR3 NM_000142 13112046 2261 D-003133-08TGCACAACCTCGACTACTA 724 FGFR4 FGFR4 NM_002011 13112051 2264 D-003134-05GCACTGGAGTCTCGTGATG 725 FGFR4 NM_002011 13112051 2264 D-003134-06CATAGGGACCTCTCGAATA 726 FGFR4 NM_002011 13112051 2264 D-003134-07ATACGGACATCATCCTGTA 727 FGFR4 NM_002011 13112051 2264 D-003134-08ATAGGGACCTCTCGAATAG 728 FGR FGR NM_005248 4885234 2268 D-003135-05GCGATCATGTGAAGCATTA 729 FGR NM_005248 4885234 2268 D-003135-06TCACTGAGCTCATCACCAA 730 FGR NM_005248 4885234 2268 D-003135-07GAAGAGTGGTACTTTGGAA 731 FGR NM_005248 4885234 2268 D-003135-08CCCAGAAGCTGCCCTCTTT 732 FLT1 FLT1 NM_002019 4503748 2321 D-003136-05GAGCAAACGTGACTTATTT 733 FLT1 NM_002019 4503748 2321 D-003136-06CCAAATGGGTTTCATGTTA 734 FLT1 NM_002019 4503748 2321 D-003136-07CAACAAGGATGCAGCACTA 735 FLT1 NM_002019 4503748 2321 D-003136-08GGACGTAACTGAAGAGGAT 736 FLT3 FLT3 NM_004119 4758395 2322 D-003137-05GAAGGCATCTACACCATTA 737 FLT3 NM_004119 4758395 2322 D-003137-06GAAGGAGTCTGGAATAGAA 738 FLT3 NM_004119 4758395 2322 D-003137-07GAATTTAAGTCGTGTGTTC 739 FLT3 NM_004119 4758395 2322 D-003137-08GGAATTCATTTCACTCTGA 740 FLT4 FLT4 NM_002020 4503752 2324 D-003138-05GCAAGAACGTGCATCTGTT 741 FLT4 NM_002020 4503752 2324 D-003138-06GCGAATACCTGTCCTACGA 742 FLT4 NM_002020 4503752 2324 D-003138-07GAAGACATTTGAGGAATTC 743 FLT4 NM_002020 4503752 2324 D-003138-08GAGCAGCCATTCATCAACA 744 FRK FRK NM_002031 4503786 2444 D-003139-05GAAACAGACTCTTCATATT 745 FRK NM_002031 4503786 2444 D-003139-06GAACAATACCACTCCAGTA 746 FRK NM_002031 4503786 2444 D-003139-07CAAGACCGGTTCCTTTCTA 747 FRK NM_002031 4503786 2444 D-003139-08GCAAGAATATCTCCAAAAT 748 FYN FYN NM_002037 23510344 2534 D-003140-05GGAATGGACTCATATGCAA 749 FYN NM_002037 23510344 2534 D-003140-06GCAGAAGAGTGGTACTTTG 750 FYN NM_002037 23510344 2534 D-003140-07CAAAGGAAG1TTACTGGAT 751 FYN NM_002037 23510344 2534 D-003140-08GAAGAGTGGTACTTTGGAA 752 HCK HCK NM_002110 4504356 3055 D-003141-05GAGATACCGTGAAACATTA 753 HCK NM_002110 4504356 3055 D-003141-06GCAGGGAGATACCGTGAAA 754 HCK NM_002110 4504356 3055 D-003141-07CATCGTGGTTGCCCTGTAT 755 HCK NM_002110 4504356 3055 D-003141-08TGTGTAAGATTGCTGACTT 756 ITK ITK NM_005546 21614549 3702 D-003144-05CAAATAATCTGGAAACCTA 757 ITK NM_005546 21614549 3702 D-003144-06GAAGAAACGAGGAATAATA 758 ITK NM_005546 21614549 3702 D-003144-07GAAACTCTCTCATCCCAAA 759 ITK NM_005546 21614549 3702 D-003144-08GGAATGGGCATGAAGGATA 760 JAK1 JAK1 NM_002227 4504802 3716 D-003145-05CCACATAGCTGATCTGAAA 761 JAK1 NM_002227 4504802 3716 D-003145-06TGAAATCACTCACATTGTA 762 JAK1 NM_002227 4504802 3716 D-003145-07TAAGGAACCTCTATCATGA 763 JAK1 NM_002227 4504802 3716 D-003145-08GCAGGTGGCTGTTAAATCT 764 JAK2 JAK2 NM_004972 13325062 3717 D-003146-05GCAAATAGATCCAGTTCTT 765 JAK2 NM_004972 13325062 3717 D-003146-06GAGCAAAGATCCAAGACTA 766 JAK2 NM_004972 13325062 3717 D-003146-07GCCAGAAACTTGAAACTTA 767 JAK2 NM_004972 13325062 3717 D-003146-08GTACAGATTTCGCAGATTT 768 JAK3 JAK3 NM_000215 4557680 3718 D-003147-05GCGCCTATCTTTCTCCTTT 769 JAK3 NM_000215 4557680 3718 D-003147-06CCAGAAATCGTAGACATTA 770 JAK3 NM_000215 4557680 3718 D-003147-07CCTCATCTCTTCAGACTAT 771 JAK3 NM_000215 4557680 3718 D-003147-08TGTACGAGCTCTTCACCTA 772 KDR KDR NM_002253 11321596 3791 D-003148-05GGAAATCTCTTGCAAGCTA 773 KDR NM_002253 11321596 3791 D-003148-06GATTACAGATCTCCATTTA 774 KDR NM_002253 11321596 3791 D-003148-07GCAGACAGATCTACGTTTG 775 KDR NM_002253 11321596 3791 D-003148-08GCGATGGCCTCTTCTGTAA 776 KIAA1079 KIAA1079 NM_014916 7662475 22853D-003149-05 GAAATTCTCTCAACTGATG 777 KIAA1079 NM_014916 7662475 22853D-003149-06 GCAGAGGTCTTCACACTTT 778 KIAA1079 NM_014916 7662475 22853D-003149-07 TAAATGATCTTCAGACAGA 779 KIAA1079 NM_014916 7662475 22853D-003149-08 GAGCAGCCCTACTCTGATA 780 KIT KIT NM_000222 4557694 3815D-003150-05 AAACACGGCTTAAGCAATT 781 KIT NM_000222 4557694 3815D-003150-06 GAACAGAACCTTCACTGAT 782 KIT NM_000222 4557694 3815D-003150-07 GGGAAGCCCTCATGTCTGA 783 KIT NM_000222 4557694 3815D-003150-08 GCAATTCCATTTATGTGTT 784 LCK LCK NM_005356 20428651 3932D-003151-05 GAACTGCCATTATCCCATA 785 LCK NM_005356 20428651 3932D-003151-06 GAGAGGTGGTGAAACATTA 786 LCK NM_005356 20428651 3932D-003151-07 GGGCCAAGTTTCCCATTAA 787 LCK NM_005356 20428651 3932D-003151-08 GCACGCTGCTCATCCGAAA 788 LTK LTK NM_002344 4505044 4058D-003152-05 TGAATTCACTCCTGCCAAT 789 LTK NM_002344 4505044 4058D-003152-06 GTGGCAACCTCAACACTGA 790 LTK NM_002344 4505044 4058D-003152-07 GGAGCTAGCTGTGGATAAC 791 LTK NM_002344 4505044 4058D-003152-08 GCAAGTTTCGCCATCAGAA 792 LYN LYN NM_002350 4505054 4067D-003153-05 GCAGATGGCTTGTGCAGAA 793 LYN NM_002350 4505054 4067D-003153-06 GGAGAAGGCTTGTATTAGT 794 LYN NM_002350 4505054 4067D-003153-07 GATGAGCTCTATGACATTA 795 LYN NM_002350 4505054 4067D-003153-08 GGTGCTAAGTTCCCTATTA 796 MATK MATK NM_002378 21450841 4145D-003154-05 TGAAGAATATCAAGTGTGA 797 MATK NM_002378 21450841 4145D-003154-06 CCGCTCAGCTCCTGCAGTT 798 MATK NM_002378 21450841 4145D-003154-07 TACTGAACCTGCAGCATTT 799 MATK NM_002378 21450841 4145D-003154-08 TGGGAGGTCTTCTCATATG 800 MERTK MERTK NM_006343 5453737 10461D-003155-05 GAACTTACCTTACATAGCT 801 MERTK NM_006343 5453737 10461D-003155-06 GGACCTGCATACTTACTTA 802 MERTK NM_006343 5453737 10461D-003155-07 TGACAGGAATCTTCTAATT 803 MERTK NM_006343 5453737 10461D-003155-08 GGTAATGGCTCAGTCATGA 804 MET MET NM_000245 4557746 4233D-003156-05 GAAAGAACCTCTCAACATT 805 MET NM_000245 4557746 4233D-003156-06 GGACAAGGCTGACCATATG 806 MET NM_000245 4557746 4233D-003156-07 CCAATGACCTGCTGAAATT 807 MET NM_000245 4557746 4233D-003156-08 GAGCATACATTAAACCAAA 808 MST1R MST1R NM_002447 4505264 4486D-003157-05 GGATGGAGCTGCTGGCTTT 809 MST1R NM_002447 4505264 4486D-003157-06 CTGCAGACCTATAGATTTA 810 MST1R NM_002447 4505264 4486D-003157-07 GCACCTGTCTCACTCTTGA 811 MST1R NM_002447 4505264 4486D-003157-08 GAAAGAGTCCATCCAGCTA 812 MUSK MUSK NM_005592 5031926 4593D-003158-05 GAAGAAGCCTCGGCAGATA 813 MUSK NM_005592 5031926 4593D-003158-06 GTAATAATCTCCATCATGT 814 MUSK NM_005592 5031926 4593D-003158-07 GGAATGAACTGAAAGTAGT 815 MUSK NM_005592 5031926 4593D-003158-08 GAGATTTCCTGGACTAGAA 816 NTRK1 NTRK1 NM_002529 4585711 4914D-003159-05 GGACAACCCTTTCGAGTTC 817 NTRK1 NM_002529 4585711 4914D-003159-06 CCAGTGACCTCAACAGGAA 818 NTRK1 NM_002529 4585711 4914D-003159-07 CCACAATACTTCAGTGATG 819 NTRK1 NM_002529 4585711 4914D-003159-08 GAAGAGTGGTCTCCGTTTC 820 NTRK2 NTRK2 NM_006180 21361305 4915D-00316D-05 GAACAGAAGTAATGAAATC 821 NTRK2 NM_006180 21361305 4915D-00316D-06 GTAATGCTGTTTCTGCTTA 822 NTRK2 NM_006180 21361305 4915D-00316D-07 GCAAGACACTCCAAGTTTG 823 NTRK2 NM_006180 21361305 4915D-00316D-08 GAAAGTCTATCACATTATC 824 NTRK3 NTRK3 NM_002530 4505474 4916D-003161-05 GAGCGAATCTGCTAGTGAA 825 NTRK3 NM_002530 4505474 4916D-003161-06 GAAGTTCACTACAGAGAGT 826 NTRK3 NM_002530 4505474 4916D-003161-07 GGTCGACGGTCCAAATTTG 827 NTRK3 NM_002530 4505474 4916D-003161-08 GAATATCACTTCCATACAC 828 PDGFRA PDGFRA NM_006206 154517875156 D-003162-05 GAAACTTCCTGGACTATTT 829 PDGFRA NM_006206 15451787 5156D-003162-06 GAGATTTGGTCAACTATTT 830 PDGFRA NM_006206 15451787 5156D-003162-07 GCACGCCGCTTCCTGATAT 831 PDGFRA NM_006206 15451787 5156D-003162-08 CATCAGAGCTGGATCTAGA 832 PDGFRB PDGFRB NM_002609 154517885159 D-003163-05 GAAAGGAGACGTCAAATAT 833 PDGFRB NM_002609 15451788 5159D-003163-06 GGAATGAGGTGGTCAACTT 834 PDGFRB NM_002609 15451788 5159D-003163-07 CAACGAGTCTCCAGTGCTA 835 PDGFRB NM_002609 15451788 5159D-003163-08 GAGAGGACCTGCCGAGCAA 836 PTK2 PTK2 NM_005607 27886592 5747D-003164-05 GAAGTTGGGTTGTCTAGAA 837 PTK2 NM_005607 27886592 5747D-003164-06 GAAGAACAATGATGTAATC 838 PTK2 NM_005607 27886592 5747D-003164-07 GGAAATTGCTTTGAAGTTG 839 PTK2 NM_005607 27886592 5747D-003164-08 GGTTCAAGCTGGATTATTT 840 PTK2B PTK2B NM_004103 27886583 2185D-003165-05 GAACATGGCTGACCTCATA 841 PTK2B NM_004103 27886583 2185D-003165-06 GGACCACGCTGCTCTATTT 842 PTK2B NM_004103 27886583 2185D-003165-07 GGACGAGGACTATTACAAA 843 PTK2B NM_004103 27886583 2185D-003165-08 TGGCAGAGCTCATCAACAA 844 PTK6 PTK6 NM_005975 27886594 5753D-003166-05 GAGAAAGTCCTGCCCGTTT 845 PTK6 NM_005975 27886594 5753D-003166-06 TGAAGAAGCTGCGGCACAA 846 PTK6 NM_005975 27886594 5753D-003166-07 CCGCGACTCTGATGAGAAA 847 PTK6 NM_005975 27886594 5753D-003166-08 TGCCCGAGCTTGTGAACTA 848 PTK7 PTK7 NM_002821 27886610 5754D-003167-05 GAGAGAAGCCCACTATTAA 849 PTK7 NM_002821 27886610 5754D-003167-06 CGAGAGAAGCCCACTATTA 850 PTK7 NM_002821 27886610 5754D-003167-07 GGAGGGAGTTGGAGATGTT 851 PTK7 NM_002821 27886610 5754D-003167-08 GAAGACATGCCGCTATTTG 852 PTK9 PTK9 NM_002822 4506274 5756D-003168-05 GAAGAACTACGACAGATTA 853 PTK9 NM_002822 4506274 5756D-003168-09 GAAGGAGACTATTTAGAGT 854 PTK9 NM_002822 4506274 5756D-003168-10 GAGCGGATGCTGTATTCTA 855 PTK9 NM_002822 4506274 5756D-003168-11 CTGCAGACTTCCTTTATGA 856 PTK9L PTK9L NM_007284 31543446 11344D-003169-05 AGAGAGAGCTCCAGCAGAT 857 PTK9L NM_007284 31543446 11344D-003169-06 TTAACGAGGTGAAGACAGA 858 PTK9L NM_007284 31543446 11344D-003169-07 ACACAGAGCCCACGGATGT 859 PTK9L NM_007284 31543446 11344D-003169-08 GCTGGGATCAGGACTATGA 860 RET RET NM_000323 21536316 5979D-003170-05 GCAAAGACCTGGAGAAGAT 861 RET NM_000323 21536316 5979D-003170-06 GCACACGGCTGCATGAGAA 862 RET NM_000323 21536316 5979D-003170-07 GAACTGGCCTGGAGAGAGT 863 RET NM_000323 21536316 5979D-003170-08 TTAAATGGATGGCAATTGA 864 ROR1 ROR1 NM_005012 4826867 4919D-003171-05 GCAAGCATCTTTACTAGGA 865 ROR1 NM_005012 4826867 4919D-003171-06 GAGCAAGGCTAAAGAGCTA 866 ROR1 NM_005012 4826867 4919D-003171-07 GAGAGCAACTTCATGTAAA 867 ROR1 NM_005012 4826867 4919D-003171-08 GAGAATGTCCTGTGTCAAA 868 ROR2 ROR2 NM_004560 19743897 4920D-003172-05 GGAACTCGCTGCTGCCTAT 869 ROR2 NM_004560 19743897 4920D-003172-06 GCAGGTGCCTCCTCAGATG 870 ROR2 NM_004560 19743897 4920D-003172-07 GCAATGTGCTAGTGTACGA 871 ROR2 NM_004560 19743897 4920D-003172-08 GAAGACAGAATATGGTTCA 872 ROS1 ROS1 NM_002944 19924164 6098D-003173-05 GAGGAGACCTTCTTACTTA 873 ROS1 NM_002944 19924164 6098D-003173-06 TTACAGAGGTTCAGGATTA 874 ROS1 NM_002944 19924164 6098D-003173-07 GAACAAACCTAAGCATGAA 875 ROS1 NM_002944 19924164 6098D-003173-08 GAAAGAGCACTTCAAATAA 876 RYK RYK NM_002958 11863158 6259D-003174-05 GAAAGATGGTTACCGAATA 877 RYK NM_002958 11863158 6259D-003174-06 CAAAGTAGATTCTGAAGTT 878 RYK NM_002958 11863158 6259D-003174-07 TCACTACGCTCTATCCTTT 879 RYK NM_002958 11863158 6259D-003174-08 GGTGAAGGATATAGCAATA 880 SRC SRC NM_005417 21361210 6714D-003175-05 GAGAACCTGGTGTGCAAAG 881 SRC NM_005417 21361210 6714D-003175-09 GAGAGAACCTGGTGTGCAA 882 SRC NM_005417 21361210 6714D-003175-10 GGAGTTTGCTGGACTTTCT 883 SRC NM_005417 21361210 6714D-003175-11 GAAAGTGAGACCACGAAAG 884 SYK SYK NM_003177 21361552 6850D-003176-05 GGAATAATCTCAAGAATCA 885 SYK NM_003177 21361552 6850D-003176-06 GAACTGGGCTCTGGTAATT 886 SYK NM_003177 21361552 6850D-003176-07 GGAAGAATCTGAGCAAATT 887 SYK NM_003177 21361552 6850D-003176-08 GAACAGACATGTCAAGGAT 888 TEC TEC NM_003215 4507428 7006D-003177-05 GAAATTGTCTAGTAAGTGA 889 TEC NM_003215 4507428 7006D-003177-06 CACCTGAAGTGTTTAATTA 890 TEC NM_003215 4507428 7006D-003177-07 GTACAAAGTCGCAATCAAA 891 TEC NM_003215 4507428 7006D-003177-08 TGGAGGAGATTCTTATTAA 892 TEK TEK NM_000459 4557868 7010D-003178-05 GAAAGAATATGCCTCCAAA 893 TEK NM_000459 4557868 7010D-003178-06 GGAATGACATCAAATTTCA 894 TEK NM_000459 4557868 7010D-003178-07 TGAAGTACCTGATATTCTA 895 TEK NM_000459 4557868 7010D-003178-08 CGAAAGACCTACGTGAATA 896 TIE TIE NM_005424 4885630 7075D-003179-05 GAGAGGAGGTTTATGTGAA 897 TIE NM_005424 4885630 7075D-003179-06 GGGACAGCCTCTACCCTTA 898 TIE NM_005424 4885630 7075D-003179-07 GAAGTTCTGTGCAAATTGG 899 TIE NM_005424 4885630 7075D-003179-08 CAACATGGCCTCAGAACTG 900 TNK1 TNK1 NM_003985 4507610 8711D-003180-05 GTTCTGGGCCTAAGTCTAA 901 TNK1 NM_003985 4507610 8711D-003180-06 GAACTGGGTCTACAAGATC 902 TNK1 NM_003985 4507610 8711D-003180-07 CGAGAGGTATCGGTCATGA 903 TNK1 NM_003985 4507610 8711D-003180-08 GGCGCATCCTGGAGCATTA 904 TXK TXK NM_003328 4507742 7294D-003181-05 GAACATCTATTGAGACAAG 905 TXK NM_003328 4507742 7294D-003181-06 TCAAGGCACTTTATGATTT 906 TXK NM_003328 4507742 7294D-003181-07 GGAGAGGAATGGCTATATT 907 TXK NM_003328 4507742 7294D-003181-08 GGATATATGTGAAGGAATG 908 TYK2 TYK2 NM_003331 4507748 7297D-003182-05 GAGGAGATCCACCACTTTA 909 TYK2 NM_003331 4507748 7297D-003182-06 GCATCCACATTGCACATAA 910 TYK2 NM_003331 4507748 7297D-003182-07 TCAAATACCTAGCCACACT 911 TYK2 NM_003331 4507748 7297D-003182-08 CAATCTTGCTGACGTCTTG 912 TYRO3 TYRO3 NM_006293 27597077 7301D-003183-05 GGTAGAAGGTGTGCCATTT 913 TYRO3 NM_006293 27597077 7301D-003183-06 ACGCTGAGATTTACAACTA 914 TYRO3 NM_006293 27597077 7301D-003183-07 GGATGGCTCCTTTGTGAAA 915 TYRO3 NM_006293 27597077 7301D-003183-08 GAGAGGAACTACGAAGATC 916 YES1 YES1 NM_005433 21071041 7525D-003184-05 GAAGGACCCTGATGAAAGA 917 YES1 NM_005433 21071041 7525D-003184-06 TAAGAAGGGTGAAAGATTT 918 YES1 NM_005433 21071041 7525D-003184-07 TCAAGAAGCTCAGATAATG 919 YES1 NM_005433 21071041 7525D-003184-08 CAGAATCCCTCCATGAATT 920

TABLE VIII Gene Locus SEQ. ID Name Acc# Gl Link Duplex # Full SequenceNO. APC2 APC2 NM_013366 7549800 29882 D-003200-05 GCAAGGACCTCTTCATCAA921 APC2 NM_013366 7549800 29882 D-003200-06 GAGAAGAAGTCCACACTAT 922APC2 NM_013366 7549800 29882 D-003200-07 GGAATGCCATCTCCCAATG 923 APC2NM_013366 7549800 29882 D-003200-09 CAACACGTGTGACATCATC 924 ATM ATMNM_000051 20336202 472 D-003201-05 GCAAGCAGCTGAAACAAAT 925 ATM NM_00005120336202 472 D-003201-06 GAATGTTGCTTTCTGAATT 926 ATM NM_000051 20336202472 D-003201-07 GACCTGAAGTCTTATTTAA 927 ATM NM_000051 20336202 472D-003201-08 AGACAGAATTCCCAAATAA 928 ATR ATR NM_001184 20143978 545D-003202-05 GAACAACACTGCTGGTTTG 929 ATR NM_001184 20143978 545D-003202-06 GAAGTCATCTGTTCATTAT 930 ATR NM_001184 20143978 545D-003202-07 GAAATAAGGTAGACTCAAT 931 ATR NM_001184 20143978 545D-003202-08 CAACATAAATCCAAGAAGA 932 BTAK BTAK NM_003600 3213196 6790D-003545-04 CAAAGAATCAGCTAGCAAA 933 BTAK NM_003600 3213196 6790D-003545-05 GAAGAGAGTTATTCATAGA 934 BTAK NM_003600 3213196 6790D-003545-07 CAAATGCCCTGTCTTACTG 935 BTAK NM_003600 3213196 6790D-003545-09 TCTCGTGACTCAGCAAATT 936 CCNA1 CCNA1 NM_003914 16306528 890D-003204-05 GAACCTGGCTAAGTACGTA 937 CCNA1 NM_003914 16306528 890D-003204-06 GCAGATCCATTCTTGAAAT 938 CCNA1 NM_003914 16306528 890D-003204-07 TCACAAGAATCAGGTGTTA 939 CCNA1 NM_003914 16306528 890D-003204-08 CATAAAGCGTACCTTGATA 940 CCNA2 CCNA2 NM_001237 16950653 890D-003205-05 GCTGTGAACTACATTGATA 941 CCNA2 NM_001237 16950653 890D-003205-06 GATGATACCTACACCAAGA 942 CCNA2 NM_001237 16950653 890D-003205-07 GCTGTTAGCCTCAAAGTTT 943 CCNA2 NM_001237 16950653 890D-003205-08 AAGCTGGCCTGAATCATTA 944 CCNB1 CCNB1 NM_031966 14327895 891D-003206-05 CAACATTACCTGTCATATA 945 CCNB1 NM_031966 14327895 891D-003206-06 CCAAATACCTGATGGAACT 946 CCNB1 NM_031966 14327895 891D-003206-07 GAAATGTACCCTCCAGAAA 947 CCNB1 NM_031966 14327895 891D-003206-08 GCACCTGGCTAAGAATGTA 948 CCNB2 CCNB2 NM_004701 10938017 9133D-003207-05 CAACAAATGTCAACAAACA 949 CCNB2 NM_004701 10938017 9133D-003207-06 GCAGCAAACTCCTGAAGAT 950 CCNB2 NM_004701 10938017 9133D-003207-07 CCAGTGATTTGGAGAATAT 951 CCNB2 NM_004701 10938017 9133D-003207-08 GTGACTACGTTAAGGATAT 952 CCNB3 CCNB3 NM_033031 14719419 85417D-003208-05 TGAACAAACTGCTGACTTT 953 CCNB3 NM_033031 14719419 85417D-003208-06 GCTAGCTGCTGCCTCCTTA 954 CCNB3 NM_033031 14719419 85417D-003208-07 CAACTCACCTCGTGTGGAT 955 CCNB3 NM_033031 14719419 85417D-003208-08 GTGGATCTCTACCTAATGA 956 CCNC CCNC NM_005190 7382485 892D-003209-05 GCAGAGCTCCCACTATTTG 957 CCNC NM_005190 7382485 892D-003209-06 GGAGTAGTTTCAAATACAA 958 CCNC NM_005190 7382485 892D-003209-07 GACCTTTGCTCCAGTATGT 959 CCNC NM_005190 7382485 892D-003209-08 GAGATTCTATGCCAGGTAT 960 CCND1 CCND1 NM_053056 16950654 595D-003210-05 TGAACAAGCTCAAGTGGAA 961 CCND1 NM_053056 16950654 595D-003210-06 CCAGAGTGATCAAGTGTGA 962 CCND1 NM_053056 16950654 595D-003210-07 GTTCGTGGCCTCTAAGATG 963 CCND1 NM_053056 16950654 595D-003210-08 CCGAGAAGCTGTGCATCTA 964 CCND2 CCND2 NM_001759 16950656 894D-003211-06 TGAATTACCTGGACCGTTT 965 CCND2 NM_001759 16950656 894D-003211-07 CGGAGAAGCTGTGCATTTA 966 CCND2 NM_001759 16950656 894D-003211-08 CTACAGACGTGCGGGATAT 967 CCND2 NM_001759 16950656 894D-003211-09 CAACACAGACGTGGATTGT 968 CCND3 CCND3 NM_001760 16950657 896D-003212-05 GGACCTGGCTGCTGTGATT 969 CCND3 NM_001760 16950657 896D-003212-06 GATTATACCTTTGCCATGT 970 CCND3 NM_001760 16950657 896D-003212-07 GACCAGCACTCCTACAGAT 971 CCND3 NM_001760 16950657 896D-003212-08 TGCGGAAGATGCTGGCTTA 972 CCNE1 CCNE1 NM_001238 17318558 898D-003213-05 GTACTGAGCTGGGCAAATA 973 CCNE1 NM_001238 17318558 898D-003213-06 GGAAATCTATCCTCCAAAG 974 CCNE1 NM_001238 17318558 898D-003213-07 GGAGGTGTGTGAAGTCTAT 975 CCNE1 NM_001238 17318558 898D-003213-08 CTAAATGACTTACATGAAG 976 CCNE2 CCNE2 NM_057749 17318564 9134D-003214-05 GGATGGAACTCATTATATT 977 CCNE2 NM_057749 17318564 9134D-003214-06 GCAGATATGTTCATGACAA 978 CCNE2 NM_057749 17318564 9134D-003214-07 CATAATATCCAGACACATA 979 CCNE2 NM_057749 17318564 9134D-003214-08 TAAGAAAGCCTCAGGTTTG 980 CCNF CCNF NM_001761 4502620 899D-003215-05 TCACAAAGCATCCATATTG 981 CCNF NM_001761 4502620 899D-003215-06 GAAGTCATGTTTACAGTGT 982 CCNF NM_001761 4502620 899D-003215-07 TAGCCTACCTCTACAATGA 983 CCNF NM_001761 4502620 899D-003215-08 GCACCCGGTTTATCAGTAA 984 CCNG1 CCNG1 NM_004060 8670528 900D-003216-05 GATAATGGCCTCAGAATGA 985 CCNG1 NM_004060 8670528 900D-003216-06 GCACGGCAATTGAAGCATA 986 CCNG1 NM_004060 8670528 900D-003216-07 GGAATAGAATGTCTTCAGA 987 CCNG1 NM_004060 8670528 900D-003216-08 TAACTCACCTTCCAACAAT 988 CCNG2 CCNG2 NM_004354 4757935 901D-003217-05 GGAGAGAGTTGGTTTCTAA 989 CCNG2 NM_004354 4757935 901D-003217-06 GGTGAAACCTAAACATTTG 990 CCNG2 NM_004354 4757935 901D-003217-07 GAAATACTGAGCCTTGATA 991 CCNG2 NM_004354 4757935 901D-003217-08 TGCCAAAGTTGAAGATTTA 992 CCNH CCNH NM_001239 17738313 902D-003218-05 GCTGATGACTTTCTTAATA 993 CCNH NM_001239 17738313 902D-003218-06 CAACTTAATTTCCACCTTA 994 CCNH NM_001239 17738313 902D-003218-07 ATACACACCTTCCCAAATT 995 CCNH NM_001239 17738313 902D-003218-08 GCTATGAAGATGATGATTA 996 CCNI CCNI NM_006835 17738314 10983D-003219-05 GCAAGCAGACCTCTACTAA 997 CCNI NM_006835 17738314 10983D-003219-07 TGAGAGAATTCCAGTACTA 998 CCNI NM_006835 17738314 10983D-003219-08 GGAATCAAACGGCTCTATA 999 CCNI NM_006835 17738314 10983D-003219-09 GAATTGGGATCTTCACACA 1000 CCNT1 CCNT1 NM_001240 17978465 904D-003220-05 TATCAACACTGCTATAGTA 1001 CCNT1 NM_001240 17978465 904D-003220-06 GAACAAACGTCCTGGTGAT 1002 CCNT1 NM_001240 17978465 904D-003220-07 GCACAAGACTCACCCATCT 1003 CCNT1 NM_001240 17978465 904D-003220-08 GCACAGACTTCTTACTTCA 1004 CCNT2A CCNT2A NM_001241 17978467905 D-003221-05 GCACAGACATCCTATTTCA 1005 CCNT2A NM_001241 17978467 905D-003221-06 GCAGGGACCTTCTATATCA 1006 CCNT2A NM_001241 17978467 905D-003221-07 GAACAGCTATATTCACAGA 1007 CCNT2A NM_001241 17978467 905D-003221-09 TTATATAGCTGCCCAGGTA 1008 CCNT2B CCNT2B NM_058241 17978468905 D-003222-05 GCACAGACATCCTATTTCA 1009 CCNT2B NM_058241 17978468 905D-003222-06 GCAGGGACCTTCTATATCA 1010 CCNT2B NM_058241 17978468 905D-003222-07 GAACAGCTATATTCACAGA 1011 CCNT2B NM_058241 17978468 905D-003222-08 GGTGAAATGTACCCAGTTA 1012 CDC16 CDC16 NM_003903 14110370 8881D-003223-05 GTAGATGGCTTGCAAGAGA 1013 CDC16 NM_003903 14110370 8881D-003223-06 TAAAGTAGCTTCACTCTCT 1014 CDC16 NM_003903 14110370 8881D-003223-07 GCTACAAGCTTACTTCTGT 1015 CDC16 NM_003903 14110370 8881D-003223-08 TGGAAGAGCCCATCAATAA 1016 CDC2 CDC2 NM_033379 27886643 983D-003552-01 GTACAGATCTCCAGAAGTA 1017 CDC2 NM_033379 27886643 983D-003552-02 GATCAACTCTTCAGGATTT 1018 CDC2 NM_033379 27886643 983D-003552-03 GGTTATATCTCATCTTTGA 1019 CDC2 NM_033379 27886643 983D-003552-04 GAACTTCGTCATCCAAATA 1020 CDC20 CDC20 NM_001255 4557436 991D-003225-05 GGGAATATATATCCTCTGT 1021 CDC20 NM_001255 4557436 991D-003225-06 GAAACGGCTTCGAAATATG 1022 CDC20 NM_001255 4557436 991D-003225-07 GAAGACCTGCCGTTACATT 1023 CDC20 NM_001255 4557436 991D-003225-08 CACCAGTGATCGACACATT 1024 CDC25A CDC25A NM_001789 4502704 993D-003226-05 GAAATTATGGCATCTGTTT 1025 CDC25A NM_001789 4502704 993D-003226-06 TACAAGGAGTTCTTTATGA 1026 CDC25A NM_001789 4502704 993D-003226-07 CCACGAGGACTTTAAAGAA 1027 CDC25A NM_001789 4502704 993D-003226-08 TGGGAAACATCAGGATTTA 1028 CDC25B CDC25B NM_004358 11641416994 D-003227-05 GCAGATACCCCTATGAATA 1029 CDC25B NM_004358 11641416 994D-003227-06 CTAGGTCGCTTCTCTCTGA 1030 CDC25B NM_004358 11641416 994D-003227-07 GAGAGCTGATTGGAGATTA 1031 CDC25B NM_004358 11641416 994D-003227-08 AAAAGGACCTCGTCATGTA 1032 CDC25C CDC25C NM_001790 12408659995 D-003228-05 GAGCAGAAGTGGCCTATAT 1033 CDC25C NM_001790 12408659 995D-003228-06 CAGAAGAGATTTCAGATGA 1034 CDC25C NM_001790 12408659 995D-003228-07 CCAGGGAGCCTTAAACTTA 1035 CDC25C NM_001790 12408659 995D-003228-08 GAAACTTGGTGGACAGTGA 1036 CDC27 CDC27 NM_001256 16554576 996D-003229-06 CATGCAAGCTGAAAGAATA 1037 CDC27 NM_001256 16554576 996D-003229-07 CAACACAAGTACCTAATCA 1038 CDC27 NM_001256 16554576 996D-003229-08 GGAGATGGATCCTAGTTAC 1039 CDC27 NM_001256 16554576 996D-003229-09 GAAAAGCCATGATGATATT 1040 CDC34 CDC34 NM_004359 16357476 997D-003230-05 GCTCAGACCTCTTCTACGA 1041 CDC34 NM_004359 16357476 997D-003230-06 GGACGAGGGCGATCTATAC 1042 CDC34 NM_004359 16357476 997D-003230-07 GATCGGGAGTACACAGACA 1043 CDC34 NM_004359 16357476 997D-003230-08 TGAACGAGCCCAACACCTT 1044 CDC37 CDC37 NM_007065 1635747811140 D-003231-05 GCGAGGAGACAGCCAATTA 1045 CDC37 NM_007065 1635747811140 D-003231-06 CACAAGACCTTCGTGGAAA 1046 CDC37 NM_007065 1635747811140 D-003231-07 ACAATCGTCATGCAATTTA 1047 CDC37 NM_007065 1635747811140 D-003231-08 GAGGAGAAATGTGCACTCA 1048 CDC45L CDC45L NM_00350434335230 8318 D-003232-05 GCACACGGATCTCCTTTGA 1049 CDC45L NM_00350434335230 8318 D-003232-06 GCAAACACCTGCTCAAGTC 1050 CDC45L NM_00350434335230 8318 D-003232-07 TGAAGAGTCTGCAAATAAA 1051 CDC45L NM_00350434335230 8318 D-003232-08 GGACGTGGATGCTCTGTGT 1052 CDC6 CDC6 NM_00125416357469 990 D-003233-05 GAACACAGCTGTCCCAGAT 1053 CDC6 NM_00125416357469 990 D-003233-06 GAGCAGAGATGTCCACTGA 1054 CDC6 NM_00125416357469 990 D-003233-07 GGAAATATCTTAGCTACTG 1055 CDC6 NM_00125416357469 990 D-003233-08 GGACGAAGATTGGTATTTG 1056 CDC7 CDC7 NM_00350311038647 8317 D-003234-05 GGAATGAGGTACCTGATGA 1057 CDC7 NM_00350311038647 8317 D-003234-06 CAGGAAAGGTGTTCACAAA 1058 CDC7 NM_00350311038647 8317 D-003234-07 CTACACAAATGCACAAATT 1059 CDC7 NM_00350311038647 8317 D-003234-08 GTACGGGAATATATGCTTA 1060 CDK10 CDK10 NM_00367432528262 8558 D-003235-05 GAACTGCTGTTGGGAACCA 1061 CDK10 NM_00367432528262 8558 D-003235-06 GGAAGCAGCCCTACAACAA 1062 CDK10 NM_00367432528262 8558 D-003235-07 GCACGCCCAGTGAGAACAT 1063 CDK10 NM_00367432528262 8558 D-003235-08 GGAAGCAGCCCTACAACAA 1064 CDK2 CDK2 NM_00179816936527 1017 D-003236-05 GAGCTTAACCATCCTAATA 1065 CDK2 NM_00179816936527 1017 D-003236-06 GAGCTTAACCATCCTAATA 1066 CDK2 NM_00179816936527 1017 D-003236-07 GTACCGAGCTCCTGAAATC 1067 CDK2 NM_00179816936527 1017 D-003236-08 GAGAGGTGGTGGCGCTTAA 1068 CDK3 CDK3 NM_0012584557438 1018 D-003237-05 GAGCATTGGTTGCATCTTT 1069 CDK3 NM_001258 45574381018 D-003237-06 GATCGGAGAGGGCACCTAT 1070 CDK3 NM_001258 4557438 1018D-003237-07 GAAGCTCTATCTGGTGTTT 1071 CDK3 NM_001258 4557438 1018D-003237-08 GCAGAGATGGTGACTCGAA 1072 CDK4 CDK4 NM_000075 456426 1019D-003238-05 GCAGCACTCTTATCTACAT 1073 CDK4 NM_000075 456426 1019D-003238-06 GGAGGAGGCCTTCCCATCA 1074 CDK4 NM_000075 456426 1019D-003238-07 TCGAAAGCCTCTCTTCTGT 1075 CDK4 NM_000075 456426 1019D-003238-08 GTACCGAGCTCCCGAAGTT 1076 CDK5 CDK5 NM_004935 4826674 1020D-003239-05 TGACCAAGCTGCCAGACTA 1077 CDK5 NM_004935 4826674 1020D-003239-06 GAGCTGAAATTGGCTGATT 1078 CDK5 NM_004935 4826674 1020D-003239-07 CAACATCCCTGGTGAACGT 1079 CDK5 NM_004935 4826674 1020D-003239-08 GGATTCCCGTCCGCTGTTA 1080 CDK6 CDK6 NM_001259 16950658 1021D-003240-05 GCAAAGACCTACTTCTGAA 1081 CDK6 NM_001259 16950658 1021D-003240-06 GAAGAAGACTGGCCTAGAG 1082 CDK6 NM_001259 16950658 1021D-003240-07 GGTCTGGACTTTCTTCATT 1083 CDK6 NM_001259 16950658 1021D-003240-08 TAACAGATATCGATGAACT 1084 CDK7 CDK7 NM_001799 16950659 1022D-003241-05 GGACATAGATCAGAAGCTA 1085 CDK7 NM_001799 16950659 1022D-003241-06 CAATAGAGCTTATACACAT 1086 CDK7 NM_001799 16950659 1022D-003241-07 CATACAAGGCTTATTCTTA 1087 CDK7 NM_001799 16950659 1022D-003241-08 GGAGACGACTTACTAGATC 1088 CDK8 CDK8 NM_001260 4502744 1024D-003242-05 CCACAGTACTCACATCAGA 1089 CDK8 NM_001260 4502744 1024D-003242-06 GCAATAACCACACTAATGG 1090 CDK8 NM_001260 4502744 1024D-003242-07 GAAGAAAGTGAGAGTTGTT 1091 CDK8 NM_001260 4502744 1024D-003242-08 GAACATGACCTCTGGCATA 1092 CDK9 CDK9 NM_0012611 7017983 1025D-003243-05 GGCCAAACGTGGACAACTA 1093 CDK9 NM_0012611 7017983 1025D-003243-06 TGACGTCCATGTTCGAGTA 1094 CDK9 NM_0012611 7017983 1025D-003243-07 CCAACCAGACGGAGTTTGA 1095 CDK9 NM_0012611 7017983 1025D-003243-08 GAAGGTGGCTCTGAAGAAG 1096 CDKN1C CDKN1C NM_000076 45574401028 D-003244-05 GACCAGAACCGCTGGGATT 1097 CDKN1C NM_000076 4557440 1028D-003244-06 GGACCGAAGTGGACAGCGA 1098 CDKN1C NM_000076 4557440 1028D-003244-08 GCAAGAGATCAGCGCCTGA 1099 CDKN1C NM_000076 4557440 1028D-003244-09 CCGCTGGGATTACGACTTC 1100 CDKN2B CDKN2B NM_004936 179816931030 D-003245-05 GCGAGGAGAACAAGGGCAT 1101 CDKN2B NM_004936 17981693 1030D-003245-06 CCAACGGAGTCAACCGTTT 1102 CDKN2B NM_004936 17981693 1030D-003245-07 CGATCCAGGTCATGATGAT 1103 CDKN2B NM_004936 17981693 1030D-003245-08 CCTGGAAGCCGGCGCGGAT 1104 CDKN2C CDKN2C NM_001262 179816971031 D-003246-05 GGACACCGCCTGTGATTTG 1105 CDKN2C NM_001262 17981697 1031D-003246-06 GCCAGGAGACTGCTACTTA 1106 CDKN2C NM_001262 17981697 1031D-003246-07 TGAAAGACCGAACTGGTTT 1107 CDKN2C NM_001262 17981697 1031D-003246-08 GAACCTGCCCTTGCACTTG 1108 CDKN2D CDKN2D NM_001800 179817001032 D-003247-05 TGGCAGTTCAAGAGGGTCA 1109 CDKN2D NM_001800 17981700 1032D-003247-06 CTCAGGACCTCGTGGACAT 1110 CDKN2D NM_001800 17981700 1032D-003247-07 TGAAGGTCCTAGTGGAGCA 1111 CDKN2D NM_001800 17981700 1032D-003247-08 AGACGGCGCTGCAGGTCAT 1112 CDT1 CDT1 NM_030928 19923847 81620D-003248-05 CCAAGGAGGCACAGAAGCA 1113 CDT1 NM_030928 19923847 81620D-003248-06 GCTTCAACGTGGATGAAGT 1114 CDT1 NM_030928 19923847 81620D-003248-07 TCTCCGGGCCAGAAGATAA 1115 CDT1 NM_030928 19923847 81620D-003248-08 GCGCAATGTTGGCCAGATC 1116 CENPA CENPA NM_001809 4585861 1058D-003249-05 GCACACACCTCTTGATAAG 1117 CENPA NM_001809 4585861 1058D-003249-06 GCAAGAGAAATATGTGTTA 1118 CENPA NM_001809 4585861 1058D-003249-07 TTACATGCAGGCCGAGTTA 1119 CENPA NM_001809 4585861 1058D-003249-08 GAGACAAGGTTGGCTAAAG 1120 CENPB CENPB NM_001810 26105977 1059D-003250-05 GGACATAGCCGCCTGCTTT 1121 CENPB NM_001810 26105977 1059D-003250-06 GCACGATCCTGAAGAACAA 1122 CENPB NM_001810 26105977 1059D-003250-07 GGAGGAGGGTGATGTTGAT 1123 CENPB NM_001810 26105977 1059D-003250-08 CCGAATGGCTGCAGAGTCT 1124 CENPC1 CENPC1 NM_001812 45027781060 D-003251-05 GCGAATAGATTATCAAGGA 1125 CENPC1 NM_001812 4502778 1060D-003251-06 GAACAGAATCCATCACAAA 1126 CENPC1 NM_001812 4502778 1060D-003251-07 CCATAAACCTCACCCAGTA 1127 CENPC1 NM_001812 4502778 1060D-003251-08 CAAGAGAACACGTTTGAAA 1128 CENPE CENPE NM_001813 4502780 1062D-003252-05 GAAGACAGCTCAAATAATA 1129 CENPE NM_001813 4502780 1062D-003252-06 CAACAAAGCTACTAAATCA 1130 CENPE NM_001813 4502780 1062D-003252-07 GGAAAGAAGTGCTACCATA 1131 CENPE NM_001813 4502780 1062D-003252-08 GGAAAGAAATGACACAGTT 1132 CENPF CENPF NM_016343 14670380 1063D-003253-05 GCGAATATCTGAATTAGAA 1133 CENPF NM_016343 14670380 1063D-003253-06 GGAAATTAATGCATCCTTA 1134 CENPF NM_016343 14670380 1063D-003253-07 GAGCGAGGCTGGTGGTTTA 1135 CENPF NM_016343 14670380 1063D-003253-08 CAAGTCATCTTTCATCTAA 1136 CENPH CENPH NM_022909 2126459064946 D-003254-05 GAAAGAAGAGATTGCAATT 1137 CENPH NM_022909 2126459064946 D-003254-06 CAGAACAAATTATGCAAGA 1138 CENPH NM_022909 2126459064946 D-003254-07 CTAGTGTGCTCATGGATAA 1139 CENPH NM_022909 2126459064946 D-003254-08 GAAACACCTATTAGAGCTA 1140 CHEK1 CHEK1 NM_00127420127419 1111 D-003255-05 CAAATTGGATGCAGACAAA 1141 CHEK1 NM_00127420127419 1111 D-003255-06 GCAACAGTATTTCGGTATA 1142 CHEK1 NM_00127420127419 1111 D-003255-07 GGACTTCTCTCCAGTAAAC 1143 CHEK1 NM_00127420127419 1111 D-003255-08 AAAGATAGATGGTACAACA 1144 CHEK2 CHEK2 NM_00719422209010 11200 D-003256-02 CTCTTACATTGCATACATA 1145 CHEK2 NM_00719422209010 11200 D-003256-03 TAAACGCCTGAAAGAAGCT 1146 CHEK2 NM_00719422209010 11200 D-003256-04 GCATAGGACTCAAGTGTCA 1147 CHEK2 NM_00719422209010 11200 D-003256-05 GAAATTGCACTGTCACTAA 1148 CNK CNK NM_0040734758015 1263 D-003257-05 GCGAGAAGATCCTAAATGA 1149 CNK NM_004073 47580151263 D-003257-07 GCAAGTGGGTTGACTACTC 1150 CNK NM_004073 4758015 1263D-003257-08 GCACATCCGTTGGCCATCA 1151 CNK NM_004073 4758015 1263D-003257-09 GACCTCAAGTTGGGAAATT 1152 CRI1 CRI1 NM_014335 7656937 23741D-003258-05 GTGATGAGATTATTGATAG 1153 CRI1 NM_014335 7656937 23741D-003258-06 GGACGAGGGCGAGGAATTT 1154 CRI1 NM_014335 7656937 23741D-003258-07 GGAAACGGAGCCTTGCTAA 1155 CRI1 NM_014335 7656937 23741D-003258-08 TCAATCGTCTGACCGAAGA 1156 E2F1 E2F1 NM_005225 12669910 1869D-003259-05 GAACAGGGCCACTGACTCT 1157 E2F1 NM_005225 12669910 1869D-003259-06 TGGACCACCTGATGAATAT 1158 E2F1 NM_005225 12669910 1869D-003259-07 CCCAGGAGGTCACTTCTGA 1159 E2F1 NM_005225 12669910 1869D-003259-08 GGCTGGACCTGGAAACTGA 1160 E2F2 E2F2 NM_004091 34485718 1870D-003260-05 GGGAGAAGACTCGGTATGA 1161 E2F2 NM_004091 34485718 1870D-003260-06 GAGGACAACCTGCAGATAT 1162 E2F2 NM_004091 34485718 1870D-003260-07 TGAAGGAGCTGATGAACAC 1163 E2F2 NM_004091 34485718 1870D-003260-08 CCAAGAAGTTCA1TTACCT 1164 E2F3 E2F3 NM_001949 12669913 1871D-003261-05 GAAATTAGATGAACTGATC 1165 E2F3 NM_001949 12669913 1871D-003261-06 TGAAGTGCCTGACTCAATA 1166 E2F3 NM_001949 12669913 1871D-003261-07 GAACAAGGCAGCAGAAGTG 1167 E2F3 NM_001949 12669913 1871D-003261-08 GAAACACACAGTCCAATGA 1168 E2F4 E2F4 NM_001950 12669914 1874D-003262-05 GGAGATTGCTGACAAACTG 1169 E2F4 NM_001950 12669914 1874D-003262-06 GAAGGTATCGGGCTAATCG 1170 E2F4 NM_001950 12669914 1874D-003262-07 GTGCAGAAGTCCAGGGAAT 1171 E2F4 NM_001950 12669914 1874D-003262-08 GGACAGTGGTGAGCTCAGT 1172 E2F5 E2F5 NM_001951 12669916 1875D-003263-05 GCAGATGACTACAACTTTA 1173 E2F5 NM_001951 12669916 1875D-003263-06 GACATCAGCTACAGATATA 1174 E2F5 NM_001951 12669916 1875D-003263-07 CAACATGTCTCTGAAAGAA 1175 E2F5 NM_001951 12669916 1875D-003263-08 GAAGACATCTGTAATTGCT 1176 E2F6 E2F6 NM_001952 12669917 1876D-003264-05 TAAACAAGGTTGCAACGAA 1177 E2F6 NM_001952 12669917 1876D-003264-06 TAGCATATGTGACCTATCA 1178 E2F6 NM_001952 12669917 1876D-003264-07 GAAACCAGATTGGATGTTC 1179 E2F6 NM_001952 12669917 1876D-003264-09 GGAACTTTCTGACTTATCA 1180 FOS FOS NM_005252 6552332 2353D-003265-05 GGGATAGCCTCTCTTACTA 1181 FOS NM_005252 6552332 2353D-003265-06 GAACAGTTATCTCCAGAAG 1182 FOS NM_005252 6552332 2353D-003265-07 GGAGACAGACCAACTAGAA 1183 FOS NM_005252 6552332 2353D-003265-08 AGACCGAGCCCTTTGATGA 1184 HIPK2 HIPK2 NM_022740 1343085928996 D-003266-06 GAGAATCACTCCAATCGAA 1185 HIPK2 NM_022740 1343085928996 D-003266-07 AGACAGGGATTAAGTCAAA 1186 HIPK2 NM_022740 1343085928996 D-003266-08 GGACAAAGACAACTAGGTT 1187 HIPK2 NM_022740 1343085928996 D-003266-09 GCACACACGTCAAATCATG 1188 HUS1 HUS1 NM_004507 310772133364 D-003267-05 ACAAAGGCCTTATGCAATA 1189 HUS1 NM_004507 31077213 3364D-003267-06 GAAGTGCACATAGATATTA 1190 HUS1 NM_004507 31077213 3364D-003267-07 AAGCTTAACTTCATCCTTT 1191 HUS1 NM_004507 31077213 3364D-003267-08 GAACTTCTTCAACGAATTT 1192 JUN JUN NM_002228 7710122 3725D-003268-05 TGGAAACGACCTTCTATGA 1193 JUN NM_002228 7710122 3725D-003268-06 GAACTGCACAGCCAGAACA 1194 JUN NM_002228 7710122 3725D-003268-07 GAGCTGGAGCGCCTGATAA 1195 JUN NM_002228 7710122 3725D-003268-08 TAACGCAGCAGTTGCAAAC 1196 JUNB JUNB NM_002229 4504808 3726D-003269-05 GCATCAAAGTGGAGCGCAA 1197 JUNB NM_002229 4504808 3726D-003269-06 TGGAAGACCAAGAGCGCAT 1198 JUNB NM_002229 4504808 3726D-003269-07 CATACACAGCTACGGGATA 1199 JUNB NM_002229 4504808 3726D-003269-08 CCATCAACATGGAAGACCA 1200 LOC51053 LOC51053 NM_01589520127542 51053 D-003270-05 GGAGAAAGGCGCTGTATGA 1201 LOC51053 NM_01589520127542 51053 D-003270-06 GAATAGTTCTGTCCCAAGA 1202 LOC51053 NM_01589520127542 51053 D-003270-07 GAACATGTACAGTATATGG 1203 LOC51053 NM_01589520127542 51053 D-003270-08 GCAGAAACAAGAAGAAATC 1204 MAD2L1 MAD2L1NM_002358 6466452 4085 D-003271-05 GAAAGATGGCAGTTTGATA 1205 MAD2L1NM_002358 6466452 4085 D-003271-06 TAAATAATGTGGTGGAACA 1206 MAD2L1NM_002358 6466452 4085 D-003271-07 GAAATCCGTTCAGTGATCA 1207 MAD2L1NM_002358 6466452 4085 D-003271-08 TTACTCGAGTGCAGAAATA 1208 MAD2L2MAD2L2 NM_006341 6006019 10459 D-003272-05 GGAAGAGCGCGCTCATAAA 1209MAD2L2 NM_006341 6006019 10459 D-003272-06 TGGAAGAGCGCGCTCATAA 1210MAD2L2 NM_006341 6006019 10459 D-003272-07 AGCCACTCCTGGAGAAGAA 1211MAD2L2 NM_006341 6006019 10459 D-003272-08 TGGAGAAATTCGTCTTTGA 1212 MCM2MCM2 NM_004526 33356546 4171 D-003273-05 GAAGATCTTTGCCAGCATT 1213 MCM2NM_004526 33356546 4171 D-003273-06 GGATAAGGCTCGTCAGATC 1214 MCM2NM_004526 33356546 4171 D-003273-07 CAGAGCAGGTGACATATCA 1215 MCM2NM_004526 33356546 4171 D-003273-08 GCCGTGGGCTCCTGTATGA 1216 MCM3 MCM3NM_002388 33356548 4172 D-003274-05 GGACATCAATATTCTTCTA 1217 MCM3NM_002388 33356548 4172 D-003274-06 GCCAGGACATCTCCAGTTA 1218 MCM3NM_002388 33356548 4172 D-003274-07 GCAGGTATGACCAGTATAA 1219 MCM3NM_002388 33356548 4172 D-003274-08 GGAAATGCCTCAAGTACAC 1220 MCM4 MCM4XM_030274 22047061 4173 D-003275-05 GGACATATCTATTCTTACT 1221 MCM4XM_030274 22047061 4173 D-003275-06 GATGTTAGTTCACCACTGA 1222 MCM4XM_030274 22047061 4173 D-003275-07 CCAGCTGCCTCATACTTTA 1223 MCM4XM_030274 22047061 4173 D-003275-08 GAAAGTACAAGATCGGTAT 1224 MCM5 MCM5NM_006739 23510447 4174 D-003276-05 GAAGATCCCTGGCATCATC 1225 MCM5NM_006739 23510447 4174 D-003276-06 GAACAGGGTTACCATCATG 1226 MCM5NM_006739 23510447 4174 D-003276-07 GGACAACATTGACTTCATG 1227 MCM5NM_006739 23510447 4174 D-003276-08 CCAAGGAGGTAGCTGATGA 1228 MCM6 MCM6NM_005915 33469920 4175 D-003277-05 GGAAAGAGCTCAGAGATGA 1229 MCM6NM_005915 33469920 4175 D-003277-06 GAGCAGCGATGGAGAAATT 1230 MCM6NM_005915 33469920 4175 D-003277-07 GGAAACACCTGATGTCAAT 1231 MCM6NM_005915 33469920 4175 D-003277-08 CCAAACATCTGCCGAAATC 1232 MCM7 MCM7NM_005916 33469967 4176 D-003278-05 GGAAATATCCCTCGTAGTA 1233 MCM7NM_005916 33469967 4176 D-003278-06 GGAAGAAGCAGTTCAAGTA 1234 MCM7NM_005916 33469967 4176 D-003278-07 CAACAAGCCTCGTGTGATC 1235 MCM7NM_005916 33469967 4176 D-003278-08 GGAGAGAACACAAGGATTG 1236 MDM2 MDM2NM_002392 4505136 4193 D-003279-05 GGAGATATGTTGTGAAAGA 1237 MDM2NM_002392 4505136 4193 D-003279-06 CCACAAATCTGATAGTATT 1238 MDM2NM_002392 4505136 4193 D-003279-07 GATGAGGTATATCAAGTTA 1239 MDM2NM_002392 4505136 4193 D-003279-08 GGAAGAAACCCAAGACAAA 1240 MK167 MK167NM_002417 19923216 4288 D-003280-05 GCACAAAGCTTGGTTATAA 1241 MK167NM_002417 19923216 4288 D-003280-06 CCTAAGACCTGAACTATTT 1242 MK167NM_002417 19923216 4288 D-003280-07 CAAAGAGGAACACAAATTA 1243 MK167NM_002417 19923216 4288 D-003280-08 GTAAATGGGTCTGTTATTG 1244 MNAT1 MNAT1NM_002431 4505224 4331 D-003281-05 GGAAGAAGCTTTAGAAGTG 1245 MNAT1NM_002431 4505224 4331 D-003281-06 TAGATGAGCTGGAGAGTTC 1246 MNAT1NM_002431 4505224 4331 D-003281-07 GGACCTTGCTGGAGGCTAT 1247 MNAT1NM_002431 4505224 4331 D-003281 -08 GCAGATAGAGACATATGGA 1248 MYC MYCNM_002467 31543215 4609 D-003282-05 CAGAGAAGCTGGCCTCCTA 1249 MYCNM_002467 31543215 4609 D-003282-06 GAAACGACGAGAACAGTTG 1250 MYCNM_002467 31543215 4609 D-003282-07 CGACGAGACCTTCATCAAA 1251 MYCNM_002467 31543215 4609 D-003282-08 CCACACATCAGCACAACTA 1252 ORC1L ORC1LNM_004153 31795543 4998 D-003283-05 GAACAGGAATTCCAAGACA 1253 ORC1LNM_004153 31795543 4998 D-003283-06 TAAGAAACGTGCTCGAGTA 1254 ORC1LNM_004153 31795543 4998 D-003283-07 GAGATCACCTCACCTTCTA 1255 ORC1LNM_004153 31795543 4998 D-003283-08 GCAGAGAGCCCTTCTTGGA 1256 ORC2L ORC2LNM_006190 32454751 4999 D-003284-05 GAAGAAACCTCCTATGAGA 1257 ORC2LNM_006190 32454751 4999 D-003284-06 GAAGGGAACTGATGGAGTA 1258 ORC2LNM_006190 32454751 4999 D-003284-07 GAAGAATGATCCTGAGATT 1259 ORC2LNM_006190 32454751 4999 D-003284-08 GAAGAGATGTTCAAGAATC 1260 ORC3L ORC3LNM_012381 32483366 23595 D-003285-05 GGACTGCTGTGTAGATATA 1261 ORC3LNM_012381 32483366 23595 D-003285-06 GAACTGATGACCATACTTG 1262 ORC3LNM_012381 32483366 23595 D-003285-07 AAAGATCTCTCTGCCAATA 1263 ORC3LNM_012381 32483366 23595 D-003285-08 CAGCACAGCTAAGAGAATA 1264 ORC4LORC4L NM_002552 32454749 5000 D-003286-06 GAAAGCACATTCCGTTTAT 1265 ORC4LNM_002552 32454749 5000 D-003286-07 TGAAAGAACTCATGGAAAT 1266 ORC4LNM_002552 32454749 5000 D-003286-08 GCTGAGAAGTGGAATGAAA 1267 ORC4LNM_002552 32454749 5000 D-003286-09 CCAGTGATCTTCATATTAG 1268 ORC5L ORC5LNM_002553 32454752 5001 D-003287-05 GAAATAACCTGTGAAACAT 1269 ORC5LNM_002553 32454752 5001 D-003287-06 CAGATTACCTCTCTAGTGA 1270 ORC5LNM_002553 32454752 5001 D-003287-07 GAACTTCCATATTACTCTA 1271 ORC5LNM_002553 32454752 5001 D-003287-08 GTATTCAGCTGATTTCTAT 1272 ORC6L ORC6LNM_014321 32454755 23594 D-003288-05 GAACATGGCTTCAAAGATA 1273 ORC6LNM_014321 32454755 23594 D-003288-06 GGACAGGGCTTATTTAATT 1274 ORC6LNM_014321 32454755 23594 D-003288-07 GAAAGAAGATAGTGGTTGA 1275 ORC6LNM_014321 32454755 23594 D-003288-08 TATCAGAGCTGTCTTAAAT 1276 PCNA PCNANM_002592 33239449 5111 D-003289-05 GATCGAGGATGAAGAAGGA 1277 PCNANM_002592 33239449 5111 D-003289-07 GCCGAGATCTCAGCCATAT 1278 PCNANM_002592 33239449 5111 D-003289-09 GAGGCCTGCTGGGATATTA 1279 PCNANM_002592 33239449 5111 D-003289-10 GTGGAGAACTTGGAAATGG 1280 PLK PLKNM_005030 21359872 5347 D-003290-05 CAACCAAAGTCGAATATGA 1281 PLKNM_005030 21359872 5347 D-003290-06 CAAGAAGAATGAATACAGT 1282 PLKNM_005030 21359872 5347 D-003290-07 GAAGATGTCCATGGAAATA 1283 PLKNM_005030 21359872 5347 D-003290-08 CAACACGCCTCATCCTCTA 1284 PIN1 PIN1NM_006221 5453897 5300 D-003291-05 GGACCAAGGAGGAGGCCCT 1285 PIN1NM_006221 5453897 5300 D-003291-06 CGTCCTGGCGGCAGGAGAA 1286 PIN1NM_006221 5453897 5300 D-003291-07 CGGGAGAGGAGGACTTTGA 1287 PIN1NM_006221 5453897 5300 D-003291-08 AGTCGGGAGAGGAGGACTT 1288 PIN1L PIN1LNM_006222 5453899 5301 D-003292-06 CGACCTGGCGGCAGGAAAT 1289 PIN1LNM_006222 5453899 5301 D-003292-07 AGGCAGGAGAGAAGGACTT 1290 PIN1LNM_006222 5453899 5301 D-003292-08 GCTACATCCAGAAGATCAA 1291 PIN1LNM_006222 5453899 5301 D-003292-09 GGACAGTGTTCACGGATTC 1292 RAD1 RAD1NM_002853 19718797 5810 D-003293-05 GAAGATGGACAAATATGTT 1293 RAD1NM_002853 19718797 5810 D-003293-06 GGAAGAGTCTGTTACTTTT 1294 RAD1NM_002853 19718797 5810 D-003293-07 GATAACAGAGGCTTCCTTT 1295 RAD1NM_002853 19718797 5810 D-003293-08 GCATTAGTCCTATCTTGTA 1296 RAD17 RAD17NM_133338 19718783 5884 D-003294-05 GAATCAAGCTTCCATATGT 1297 RAD17NM_133338 19718783 5884 D-003294-06 CAACAAAGCCCGAGGATAT 1298 RAD17NM_133338 19718783 5884 D-003294-07 ACACATGCCTGGAGACTTA 1299 RAD17NM_133338 19718783 5884 D-003294-08 CTACATAGATTTCTTCATG 1300 RAD9A RAD9ANM_004584 19924112 5883 D-003295-05 TCAGCAAACTTGAATCTTA 1301 RAD9ANM_004584 19924112 5883 D-003295-06 GACATTGACTCTTACATGA 1302 RAD9ANM_004584 19924112 5883 D-003295-08 GGAAACCACTATAGGCAAT 1303 RAD9ANM_004584 19924112 5883 D-003295-09 CGGACGACTTTGCCAATGA 1304 RB1 RB1NM_000321 19924112 5925 D-003296-05 GAAAGGACATGTGAACTTA 1305 RB1NM_000321 19924112 5925 D-003296-06 GAAGAAGTATGATGTATTG 1306 RB1NM_000321 4506434 5925 D-003296-07 GAAATGACTTCTACTCGAA 1307 RB1NM_000321 4506434 5925 D-003296-08 GGAGGGAACATCTATATTT 1308 RBBP2 RBBP2NM_005056 4826967 5927 D-003297-05 CAAAGAAGCTGAATAAACT 1309 RBBP2NM_005056 4826967 5927 D-003297-06 CAACACATATGGCGGATTT 1310 RBBP2NM_005056 4826967 5927 D-003297-07 GGACAAACCTAGAAAGAAG 1311 RBBP2NM_005056 4826967 5927 D-003297-08 GAAAGGCACTCTCTCTGTT 1312 RBL1 RBL1NM_002895 34577078 5933 D-003298-05 CAAGAGAAGTTGTGGCATA 1313 RBL1NM_002895 34577078 5933 D-003298-06 CAGCAGCACTCCATTTATA 1314 RBL1NM_002895 34577078 5933 D-003298-07 ACAGAAAGGTCTATCATTT 1315 RBL1NM_002895 34577078 5933 D-003298-08 GGACATAAAGTTACAATTC 1316 RBL2 RBL2NM_005611 21361291 5934 D-003299-05 GAGCAGAGCTTAATCGAAT 1317 RBL2NM_005611 21361291 5934 D-003299-06 GAGAATAGCCCTTGTGTGA 1318 RBL2NM_005611 21361291 5934 D-003299-07 GGACTTAGTTTATGGAAAT 1319 RBL2NM_005611 21361291 5934 D-003299-08 GAATTTAGATGAGCGGATA 1320 RBP1 RBP1NM_002899 8400726 5947 D-003300-05 GAGACAAGCTCCAGTGTGT 1321 RBP1NM_002899 8400726 5947 D-003300-06 GCAAGCAAGTATTCAAGAA 1322 RBP1NM_002899 8400726 5947 D-003300-07 GCAGGACGGTGACCATATG 1323 RBP1NM_002899 8400726 5947 D-003300-08 GCAAGTGCATGACAACAGT 1324 RPA3 RPA3NM_002947 19923751 6119 D-003322-05 GGAAGTGGTTGGAAGAGTA 1325 RPA3NM_002947 19923751 6119 D-003322-06 GAAGATAGCCATCCTTTTG 1326 RPA3NM_002947 19923751 6119 D-003322-07 CATGCTAGCTCAATTCATC 1327 RPA3NM_002947 19923751 6119 D-003322-08 GATCTTGGACTTTACAATG 1328 SKP1A SKP1ANM_006930 25777710 6500 D-003323-05 GGAGAGATATTTGAAGTTG 1329 SKP1ANM_006930 25777710 6500 D-003323-06 GGGAATGGATGATGAAGGA 1330 SKP1ANM_006930 25777710 6500 D-003323-07 CAAACAATCTGTGACTATT 1331 SKP1ANM_006930 25777710 6500 D-003323-08 TCAATTAAGTTGCAGAGTT 1332 SKP2 SKP2NM_005983 16306594 6502 D-003324-05 CATCTAGACTTAAGTGATA 1333 SKP2NM_005983 16306594 6502 D-003324-06 GAAATCAGATCTCTCTACT 1334 SKP2NM_005983 16306594 6502 D-003324-07 CTAAAGGTCTCTGGTGTTT 1335 SKP2NM_005983 16306594 6502 D-003324-08 GATGGTACCC1TCAACTGT 1336 SNK SNKNM_006622 5730054 10769 D-003325-05 GAAGACATCTACAAGCTTA 1337 SNKNM_006622 5730054 10769 D-003325-06 GAAATACCTTCATGAACAA 1338 SNKNM_006622 5730054 10769 D-003325-07 GAAGGTCAATGGCTCATAT 1339 SNKNM_006622 5730054 10769 D-003325-08 CCGGAGATCTCGCGGATTA 1340 STK12 STK12NM_004217 4759177 9212 D-003326-07 CAGAAGAGCTGCACATTTG 1341 STK12NM_004217 4759177 9212 D-003326-08 CCAAACTGCTCAGGCATAA 1342 STK12NM_004217 4759177 9212 D-003326-09 ACGCGGCACTTCACAATTG 1343 STK12NM_004217 4759177 9212 D-003326-10 TGGGACACCCGACATCTTA 1344 TFDP1 TFDP1NM_007111 34147667 7027 D-003327-05 GGAAGCAGCTCTTGCCAAA 1345 TFDP1NM_007111 34147667 7027 D-003327-06 GAGGAGACTTGAAAGAATA 1346 TFDP1NM_007111 34147667 7027 D-003327-07 GAACTTAGAGGTGGAAAGA 1347 TFDP1NM_007111 34147667 7027 D-003327-08 GCGAGAAGGTGCAGAGGAA 1348 TFDP2 TFDP2NM_006286 5454111 7029 D-003328-05 GAAAGTGTGTGAGAAAGTT 1349 TFDP2NM_006286 5454111 7029 D-003328-06 CACAGGACCTTCTTGGTTA 1350 TFDP2NM_006286 5454111 7029 D-003328-07 CGAAATCCCTGGTGCCAAA 1351 TFDP2NM_006286 5454111 7029 D-003328-08 TGAGATCCATGATGACATA 1352 TP53 TP53NM_000546 8400737 7157 D-003329-05 GAGGTTGGCTCTGACTGTA 1353 TP53NM_000546 8400737 7157 D-003329-06 CAGTCTACCTCCCGCCATA 1354 TP53NM_000546 8400737 7157 D-003329-07 GCACAGAGGAAGAGAATCT 1355 TP53NM_000546 8400737 7157 D-003329-08 GAAGAAACCACTGGATGGA 1356 TP63 TP63NM_003722 31543817 8626 D-003330-05 CATCATGTCTGGACTATTT 1357 TP63NM_003722 31543817 8626 D-003330-06 CAAACAAGATTGAGATTAG 1358 TP63NM_003722 31543817 8626 D-003330-07 GCACACAGACAAATGAATT 1359 TP63NM_003722 31543817 8626 D-003330-08 CGACAGTCTTGTACAATTT 1360 TP73 TP73NM_005427 4885644 7161 D-003331-05 GCAAGCAGCCCATCAAGGA 1361 TP73NM_005427 4885644 7161 D-003331-06 GAGACGAGGACACGTACTA 1362 TP73NM_005427 4885644 7161 D-003331-07 CTGCAGAACCTGACCATTG 1363 TP73NM_005427 4885644 7161 D-003331-08 GGCCATGCCTGTTTACAAG 1364 YWHAZ YWHAZNM_003406 21735623 7534 D-003332-05 GCAAGGAGCTGAATTATCC 1365 YWHAZNM_003406 21735623 7534 D-003332-06 TAAGAGATATCTGCAATGA 1366 YWHAZNM_003406 21735623 7534 D-003332-07 GACGGAAGGTGCTGAGAAA 1367 YWHAZNM_003406 21735623 7534 D-003332-08 AGAGCAAAGTCTTCTATTT 1368

TABLE IX Gene SEQ. ID Name Accession # GI# Duplex # Sequence NO. ARNM_000044 21322251 D-003400-01 GGAACTCGATCGTATCATT 1369 AR NM_00004421322251 D-003400-02 CAAGGGAGGTTACACCAAA 1370 AR NM_000044 21322251D-003400-03 TCAAGGAACTCGATCGTAT 1371 AR NM_000044 21322251 D-003400-04GAAATGATTGCACTATTGA 1372 ESR1 NM_000125 4503602 D-003401-01GAATGTGCCTGGCTAGAGA 1373 ESR1 NM_000125 4503602 D-003401-02CATGAGAGCTGCCAACCTT 1374 ESR1 NM_000125 4503602 D-003401-03AGAGAAAGATTGGCCAGTA 1375 ESR1 NM_000125 4503602 D-003401-04CAAGGAGACTCGCTACTGT 1376 ESR2 NM_001437 10835012 D-003402-01GAACATCTGCTCAACATGA 1377 ESR2 NM_001437 10835012 D-003402-02GCACGGCTCCATATACATA 1378 ESR2 NM_001437 10835012 D-003402-03CAAGAAGATTCCCGGCTTT 1379 ESR2 NM_001437 10835012 D-003402-04GGAAATGCGTAGAAGGAAT 1380 ESRRA NM_004451 18860919 D-003403-01GGCCTTCGCTGAGGACTTA 1381 ESRRA NM_004451 18860919 D-003403-02TGAATGCACTGGTGTCTCA 1382 ESRRA NM_004451 18860919 D-003403-03GCATTGAGCCTCTCTACAT 1383 ESRRA NM_004451 18860919 D-003403-04CCAGACAGCGGGCAAAGTG 1384 ESRRB NM_004452 22035686 D-003404-01TACCTGAGCTTACAAATTT 1385 ESRRB NM_004452 22035686 D-003404-02GCACTTCTATAGCGTCAAA 1386 ESRRB NM_004452 22035686 D-003404-03CAACTCCGATTCCATGTAC 1387 ESRRB NM_004452 22035686 D-003404-04GGACTCGCCACCCATGTTT 1388 ESRRG NM_001438 4503604 D-003405-01AAACAAAGATCGACACATT 1389 ESRRG NM_001438 4503604 D-003405-02TCAGGAAACTGTATGATGA 1390 ESRRG NM_001438 4503604 D-003405-03GAAGACCAGTCCAAATTAG 1391 ESRRG NM_001438 4503604 D-003405-04ATGAAGCGCTGCAGGATTA 1392 HNF4A NM_000457 21361184 D-003406-01CGACATCACTGGAGCATAT 1393 HNF4A NM_000457 21361184 D-003406-02GAAGGAAGCCGTCCAGAAT 1394 HNF4A NM_000457 21361184 D-003406-03CCAAGTACATCCCAGCTTT 1395 HNF4A NM_000457 21361184 D-003406-04GGACATGGCCGACTACAGT 1396 HNF4G NM_004133 6631087 D-003407-01GCACTGACATAAACGTTAA 1397 HNF4G NM_004133 6631087 D-003407-02ACAAAGAGATCCATGATGT 1398 HNF4G NM_004133 6631087 D-003407-03AGAGATCCATGATGTATAA 1399 HNF4G NM_004133 6631087 D-003407-04AAATGAACGTGACAGAATA 1400 H5AJ2425 NM_017532 8923776 D-003408-01GAATGAATCTACACCTTTG 1401 H5AJ2425 NM_017532 8923776 D-003408-02GGAAATACGTGGAGACACT 1402 H5AJ2425 NM_017532 8923776 D-003408-03CCAGATAACTACGGCGATA 1403 H5AJ2425 NM_017532 8923776 D-003408-04TGGCGTACCTTCTCATTGA 1404 NROB1 NM_000475 5016089 D-003409-01CAGCATGGATGATATGATG 1405 NROB1 NM_000475 5016089 D-003409-02CTGCTGAGATTCATCAATG 1406 NROB1 NM_000475 5016089 D-003409-03ACAGATTCATCGAACTTAA 1407 NROB1 NM_000475 5016089 D-003409-04GAACGTGGCGCTCCTGTAC 1408 NROB2 NM_021969 13259502 D-003410-01GAATATGCCTGCCTGAAAG 1409 NROB2 NM_021969 13259502 D-003410-02GGAATATGCCTGCCTGAAA 1410 NROB2 NM_021969 13259502 D-003410-03CGTAGCCGCTGCCTATGTA 1411 NROB2 NM_021969 13259502 D-003410-04GCCATTCTCTACGCACTTC 1412 NR1D1 NM_021724 13430847 D-003411-01CAACACAGGTGGCGTCATC 1413 NR1D1 NM_021724 13430847 D-003411-02GGCATGGTGTTACTGTGTA 1414 NR1D1 NM_021724 13430847 D-003411-03CAACATGCATTCCGAGAAG 1415 NR1D1 NM_021724 13430847 D-003411-04GCGCTTTGCTTCGTTGTTC 1416 NR1H2 NM_007121 11321629 D-003412-01GAACAGATCCGGAAGAAGA 1417 NR1H2 NM_007121 11321629 D-003412-02GAAGAACAGATCCGGAAGA 1418 NR1H2 NM_007121 11321629 D-003412-03CTAAGCAAGTGCCTGGTTT 1419 NR1H2 NM_007121 11321629 D-003412-04GCTAACAGCGGCTCAAGAA 1420 NR1H3 NM_005693 5031892 D-003413-01GAACAGATCCGCCTGAAGA 1421 NR1H3 NM_005693 5031892 D-003413-02GGAGATAGTTGACTTTGCT 1422 NR1H3 NM_005693 5031892 D-003413-03GAGTTTGCCTTGCTCATTG 1423 NR1H3 NM_005693 5031892 D-003413-04TGACT1TGCTAAACAGCTA 1424 NR1H4 NM_005123 4826979 D-003414-01CAAGTGACCTCGACAACAA 1425 NR1H4 NM_005123 4826979 D-003414-02GAAAGAATTCGAAATAGTG 1426 NR1H4 NM_005123 4826979 D-003414-03CAACAGACTCTTCTACATT 1427 NR1H4 NM_005123 4826979 D-003414-04GAACCATACTCGCAATACA 1428 NR1I2 NM_003889 11863133 D-003415-01GAACCATGCTGACTTTGTA 1429 NR1I2 NM_003889 11863133 D-003415-02GATGGACGCTCAGATGAAA 1430 NR1I2 NM_003889 11863133 D-003415-03CAACCTACATGTTCAAAGG 1431 NR1I2 NM_003889 11863133 D-003415-04CAGGAGCAATTCGCCATTA 1432 NR1I3 NM_005122 4826660 D-003416-01GGAAATCTGTCACATCGTA 1433 NR1I3 NM_005122 4826660 D-003416-02TCGCAGACATCAACACTTT 1434 NR1I3 NM_005122 4826660 D-003416-03CCTCTTCGCTACACAATTG 1435 NR1I3 NM_005122 4826660 D-003416-04GAACAGTTTGTGCAGTTTA 1436 NR2C1 NM_003297 4507672 D-003417-01TGACAGCACTTGATCATAA 1437 NR2C1 NM_003297 4507672 D-003417-02GGAAGGAAGTGTACACCTA 1438 NR2C1 NM_003297 4507672 D-003417-03GAGCACATCTTCAAACTAC 1439 NR2C1 NM_003297 4507672 D-003417-04GAAGAAATTGCACATCAAA 1440 NR2C2 NM_003298 4507674 D-003418-01GAACAACGGTGACACTTCA 1441 NR2C2 NM_003298 4507674 D-003418-02CTGATGAGCTCCAACATAA 1442 NR2C2 NM_003298 4507674 D-003418-03CAACCTAAGTGAATCTTTG 1443 NR2C2 NM_003298 4507674 D-003418-04GAAGACACCTACCGATTGG 1444 NR2E1 NM_003269 21361108 D-003419-01GATCATATCTGAAATACAG 1445 NR2E1 NM_003269 21361108 D-003419-02CAAGACTGCTTTCAGATAT 1446 NR2E1 NM_003269 21361108 D-003419-03GTTAGATGCTACTGAATTT 1447 NR2E1 NM_003269 21361108 D-003419-04CAATGTATCTCTATGAAGT 1448 NR2E3 NM_014249 7657394 D-003420-01GAGAAGCTCCTTTGTGATA 1449 NR2E3 NM_014249 7657394 D-003420-02GAAGCACTATGGCATCTAT 1450 NR2E3 NM_014249 7657394 D-003420-03GAAGGATCCTGAGCACGTA 1451 NR2E3 NM_014249 7657394 D-003420-04GAAGCTCCTTTGTGATATG 1452 NR2F1 NM_005654 20127484 D-003421-01GAAACTCTCATCCGCGATA 1453 NR2F1 NM_005654 20127484 D-003421-02TCTCATCCGCGATATGTTA 1454 NR2F1 NM_005654 20127484 D-003421-03CAAGAAGTGCCTCAAAGTG 1455 NR2F1 NM_005654 20127484 D-003421-04GGAACTTAACTTACACATG 1456 NR2F2 NM_021005 14149745 D-003422-01GTACCTGTCCGGATATATT 1457 NR2F2 NM_021005 14149745 D-003422-02CCAACCAGCCGACGAGATT 1458 NR2F2 NM_021005 14149745 D-003422-03ACTCGTACCTGTCCGGATA 1459 NR2F2 NM_021005 14149745 D-003422-04GGCCGTATATGGCAATTCA 1460 NR2F6 NM_005234 20070198 D-003423-01CGACGCCTGTGGCCTCTCA 1461 NR2F6 NM_005234 20070198 D-003423-02CAGCCGGTGTCCGAACTGA 1462 NR2F6 NM_005234 20070198 D-003423-03CAACCGTGACTGCCAGATC 1463 NR2F6 NM_005234 20070198 D-003423-04GTACTGCCGTCTCAAGAAG 1464 NR3C1 NM_000176 4504132 D-003424-01GAGGACAGATGTACCACTA 1465 NR3C1 NM_000176 4504132 D-003424-02GATAAGACCATGAGTATTG 1466 NR3C1 NM_000176 4504132 D-003424-03GAAGACGATTCATTCCTTT 1467 NR3C1 NM_000176 4504132 D-003424-04GGACAGATGTACCACTATG 1468 NR3C2 NM_000901 4505198 D-003425-01GCAAACAGATGATCCAAGT 1469 NR3C2 NM_000901 4505198 D-003425-02CAGCTAAGATTTATCAGAA 1470 NR3C2 NM_000901 4505198 D-003425-03GCACGAAAGTCAAAGAAGT 1471 NR3C2 NM_000901 4505198 D-003425-04GGTATCCGGTCTTAGAATA 1472 NR4A1 NM_002135 21361341 D-003426-01GAAGGAAGTTGTCCGAACA 1473 NR4A1 NM_002135 21361341 D-003426-02CAGGAGAGTTTGACACCTT 1474 NR4A1 NM_002135 21361341 D-003426-03CAGTGGCTCTGACTACTAT 1475 NR4A1 NM_002135 21361341 D-003426-04GAAGGCCGCTGTGCTGTGT 1476 NR4A2 NM_006186 5453821 D-003427-01GCAATGCGTTCGTGGCTTT 1477 NR4A2 NM_006186 5453821 D-003427-02CGGCTACACAGGAGAGTTT 1478 NR4A2 NM_006186 5453821 D-003427-03CCACGTGACTTTCAACAAT 1479 NR4A2 NM_006186 5453821 D-003427-04GAATACAGCTCCGATTTCT 1480 NR4A3 NM_006981 11276070 D-003428-01CAAAGAAGATCAGACATTA 1481 NR4A3 NM_006981 11276070 D-003428-02GATCAGACATTACTTATTG 1482 NR4A3 NM_006981 11276070 D-003428-03CCAGAGATCTTGATTATTC 1483 NR4A3 NM_006981 11276070 D-003428-04GAAGTTGTCCGTACAGATA 1484 NR5A1 NM_004959 20070192 D-003429-01GATTTGAAGTTCCTGAATA 1485 NR5A1 NM_004959 20070192 D-003429-02GGAGCGAGCTGCTGGTGTT 1486 NR5A1 NM_004959 20070192 D-003429-03GGAGGTGGCCGACCAGATG 1487 NR5A1 NM_004959 20070192 D-003429-04CAACGTGCCTGAGCTCATC 1488 NR5A2 NM_003822 20070161 D-003430-01CCAAACATATGGCCACTTT 1489 NR5A2 NM_003822 20070161 D-003430-02TCAGAGAACTTAAGGTTGA 1490 NR5A2 NM_003822 20070161 D-003430-03GGATCCATCTTCCTGGTTA 1491 NR5A2 NM_003822 20070161 D-003430-04AAGAATACCTCTACTACAA 1492 NR6A1 NM_033334 15451847 D-003431-01CAACGAACCTGTCTCATTT 1493 NR6A1 NM_033334 15451847 D-003431-02GAAGAACTACACAGATTTA 1494 NR6A1 NM_033334 15451847 D-003431-03GAAGATGGATACGCTGTGA 1495 NR6A1 NM_033334 15451847 D-003431-04AAACGATACTGGTACATTT 1496 null D16815 2116671 D-003432-01GAAGAATGATCGAATAGAT 1497 null D16815 2116671 D-003432-02GAACATGGAGCAATATAAT 1498 null D16815 2116671 D-003432-03GAGGAGCTCTTGGCCTTTA 1499 null D16815 2116671 D-003432-04TAAACAACATGCACTCTGA 1500 PGR NM_000926 4505766 D-003433-01GAGATGAGGTCAAGCTACA 1501 PGR NM_000926 4505766 D-003433-02CAGCGTTTCTATCAACTTA 1502 PGR NM_000926 4505766 D-003433-03AGATAACTCTCATTCAGTA 1503 PGR NM_000926 4505766 D-003433-04GTAGTCAAGTGGTCTAAAT 1504 PPARA NM_005036 7549810 D-003434-01TCACGGAGCTCACGGAATT 1505 PPARA NM_005036 7549810 D-003434-02GAACATGACATAGAAGATT 1506 PPARA NM_005036 7549810 D-003434-03GGATAGTTCTGGAAGCTTT 1507 PPARA NM_005036 7549810 D-003434-04GACTCAAGCTGGTGTATGA 1508 PPARD NM_006238 5453939 D-003435-01GAGCGCAGCTGCAAGATTC 1509 PPARD NM_006238 5453939 D-003435-02GCATGAAGCTGGAGTACGA 1510 PPARD NM_006238 5453939 D-003435-03GGAAGCAGTTGGTGAATGG 1511 PPARD NM_006238 5453939 D-003435-04GCTGCAAGATTCAGAAGAA 1512 PPARG NM_138712 20336234 D-003436-01AGACTCAGCTCTACAATAA 1513 PPARG NM_138712 20336234 D-003436-02GATTGAAGCTTATCTATGA 1514 PPARG NM_138712 20336234 D-003436-03AAGTAACTCTCCTCAAATA 1515 PPARG NM_138712 20336234 D-003436-04GCATTTCTACTCCACATTA 1516 RARA NM_000964 4506418 D-003437-01GACAAGAACTGCATCATCA 1517 RARA NM_000964 4506418 D-003437-02GCAAATACACTACGAACAA 1518 RARA NM_000964 4506418 D-003437-03GAACAACAGCTCAGAACAA 1519 RARA NM_000964 4506418 D-003437-04GAGCAGCAGTTCTGAAGAG 1520 RARB NM_000965 14916493 D-003438-01GCACACTGCTCAATCAATT 1521 RARB NM_000965 14916493 D-003438-02GCAGAAGTATTCAGAAGAA 1522 RARB NM_000965 14916493 D-003438-03GGAATGACAGGAACAAGAA 1523 RARB NM_000965 14916493 D-003438-04GCACAGTCCTAGCATCTCA 1524 RARG NM_000966 21359851 D-003439-01GAAATGACCGGAACAAGAA 1525 RARG NM_000966 21359851 D-003439-02TAGAAGAGCTCATCACCAA 1526 RARG NM_000966 21359851 D-003439-03CAAGGAAGCTGTGCGAAAT 1527 RARG NM_000966 21359851 D-003439-04TCAGTGAGCTGGCTACCAA 1528 RORA NM_134261 19743902 D-003440-01GGAAAGAGTTTATGTTCTA 1529 RORA NM_134261 19743902 D-003440-02CAAGATCTGTGGAGACAAA 1530 RORA NM_134261 19743902 D-003440-03GCACCTGACTGAAGATGAA 1531 RORA NM_134261 19743902 D-003440-04CCGAGAAGATGGAATACTA 1532 RORB NM_006914 19743906 D-003441-01GCACAGAACATCATTAAGT 1533 RORB NM_006914 19743906 D-003441-02CCACACCTATGAAGAAATT 1534 RORB NM_006914 19743906 D-003441-03GATCAAATTCTACTTCTGA 1535 RORB NM_006914 19743906 D-003441-04TCAAACAGATAAAGCAAGA 1536 RORC NM_005060 19743908 D-003442-01TAGAACAGCTGCAGTACAA 1537 RORC NM_005060 19743908 D-003442-02TCACCGAGGCCATTCAGTA 1538 RORC NM_005060 19743908 D-003442-03GAACAGCTGCAGTACAATC 1539 RORC NM_005060 19743908 D-003442-04CCTCATGCCACCTTGAATA 1540 RXRA NM_002957 21536318 D-003443-01TGACGGAGCTTGTGTCCAA 1541 RXRA NM_002957 21536318 D-003443-02CAACAAGGACTGCCTGATT 1542 RXRA NM_002957 21536318 D-003443-03GCAAGGACCTGACCTACAC 1543 RXRA NM_002957 21536318 D-003443-04GCAAGGACCGGAACGAGAA 1544 RXRB NM_021976 21687229 D-003444-01GCAAAGACCTTACATACTC 1545 RXRB NM_021976 21687229 D-003444-02GCAATCATTCTGTTTAATC 1546 RXRB NM_021976 21687229 D-003444-03TCACACCGATCCATTGATG 1547 RXRB NM_021976 21687229 D-003444-04GCAAACGGCTATGTGCAAT 1548 RXRG NM_006917 21361386 D-003445-01GGAAGGACCTCATCTACAC 1549 RXRG NM_006917 21361386 D-003445-02GCGGATCTCTGGTTAAACA 1550 RXRG NM_006917 21361386 D-003445-03GCGAGCCATTGTACTCTTT 1551 RXRG NM_006917 21361386 D-003445-04GAGCCATTGTACTCTTTAA 1552 THRA NM_003250 20127451 D-003446-01GGACAAAGACGAGCAGTGT 1553 THRA NM_003250 20127451 D-003446-02GGAAACAGAGGCGGAAATT 1554 THRA NM_003250 20127451 D-003446-03GTAAGCTGATTGAGCAGAA 1555 THRA NM_003250 20127451 D-003446-04GAACCTCCATCCCACCTAT 1556 THRB NM_000461 10835122 D-003447-01GAATGTCGCTTTAAGAAAT 1557 THRB NM_000461 10835122 D-003447-02GAACAGTCGTCGCCACATC 1558 THRB NM_000461 10835122 D-003447-03GGACAAGCACCAATAGTCA 1559 THRB NM_000461 10835122 D-003447-04GTGGAAAGGTTGACTTGGA 1560 VDR NM_000376 4507882 D-003448-01TGAAGAAGCTGAACTTGCA 1561 VDR NM_000376 4507882 D-003448-02GCAACCAAGACTACAAGTA 1562 VDR NM_000376 4507882 D-003448-03TCAATGCTATGACCTGTGA 1563 VDR NM_000376 4507882 D-003448-04CCATTGAGGTCATCATGTT 1564

TABLE X Gene Symbol Sense SEQ. ID NO. ABCB1 GACCAUAAAUGUAAGGUUU 1565UAGAAGAUCUGAUGUCAAA 1566 GAAAUGUUCACUUCAGUUA 1567 GAAGAUCGCUACUGAAGCA1568 ABCC1 GGAAGCAACUGCAGAGACA 1569 GAUGACACCUCUCAACAAA 1570UAAAGUUGCUCAUCAAGUU 1571 CAACGAGUCUGCCGAAGGA 1572 ABCG2GCAGAUGCCUUCUUCGUUA 1573 AGGCAAAUCUUCGUUAUUA 1574 GGGAAGAAAUCUGGUCUAA1575 UGACUCAUCCCAACAUUUA 1576 KCNH2 CCGACGUGCUGCCUGAGUA 1577GAGAAGAGCAGCGACACUU 1578 GAUCAUAGCACCUAAGAUA 1579 GCUAUUUACUGCUCUUAUU1580 UCACUGGGCUCCUUUAAUU 1581 GUGCGAGCCUUCUGAAUAU 1582GCUAAGCUAUACUACUGUA 1583 UGACGGCGCUCUACUUCAC 1584 KCNH1GAGAUGAAUUCCUUUGAAA 1585 GAAGAACGCAUGAAACGAA 1586 GAUAAAGACACGAUUGAAA1587 GCUGAGAGGUCUAUUUAAA 1588 CLCA1 GAACAACAAUGGCUAUGAA 1589GUACAUACCUGGCUGGAUU 1590 GAACAGCUCACAAGUAUAU 1591 GGAAACGUGUGUCUAUAUU1592 SLC6A1 GGAGGUGGGAGGACAGUUA 1593 UCACAGCCCUGGUGGAUGA 1594GAAGCUGGCUCCUAUGUUC 1595 GGUCAACACUACCAACAUG 1596 SLC6A2GAACACAAGGUCAACAUUG 1597 AGAAGGAGCUGGCCUAGUG 1598 CGGAAACUCUUCACAUUUG1599 CAACAAAUUUGACAACAAC 1600 SLC21A2 GUACAUCUCCAUCUUAUUU 1601GGAAGUGGCUGAGUUAUUA 1602 GAAGGGAGGCUCAAUGUAA 1603 GAAGGAAGUGGCUGAGUUA1604 SLC21A3 GUAGAAACAGGAGCUAUUA 1605 CAAGAUUACUGUCAAACAA 1606GCACAAGAGUAUUUGGUAA 1607 GCAAAUGUCCCUUCUGUAU 1608 GCAUGACUCCUAUAUAAUA1609 AAACAGCAAUUUCCCUUAA 1610 GAAAAUGCCUCUUCAGGAA 1611 SLC28A1GUUCAUCGCUCUCCUCUUU 1612 GGAUCAAGCUGUUUCUGAA 1613 GGACUGCAGUUUGUACUUG1614 GAGUGAAACUGACCUAUGG 1615 SLC29A1 GAACGCUGCUCCCGUGGAA 1616GAAAGCCACUCUAUCAAAG 1617 GAAACCAGGUGCCUUCAGA 1618 CCUCACAGCUGUAUUCAUG1619 SLC26A1 CCACGGAGCUGCUGGUCAU 1620 GGGUUGACAUCUUAUUUGA 1621GCACGAGGGUCUCUGUGUU 1622 GGCCAUCGCCUACUCAUUG 1623 CAACACCCAUGGCAAUUAA1624 GAGGAAAGAUCUUGCUGAU 1625 GAGCAAGCGUCCUCCAAAU 1626GCAACACCCAUGGCAAUUA 1627 SLC26A2 CCAAAGAACUCAAUGAACA 1628ACAAGAACCUUCAGACUAA 1629 GAAGGUAGAUAGAAGAAUG 1630 GUAUUGAACUGUACUGUAA1631 SLC4A4 GCAAUUCUCUUCAUUUAUC 1632 GGAAAGAUGUCCACUGAAA 1633GGACAAAGCCUUCUUCAAU 1634 GGAAUGGGAUCCAGCAAUU 1635 GLRA1UGAAAGCCAUUGACAUUUG 1636 CAGACACGCUGGAGUUUAA 1637 CAAUAGCGCUUUCUGGUUU1638 GCAGGUAGCAGAUGGACUA 1639 KLK1 UCAGAGUGCUGUCUUAUGU 1640CAACUUGUUUGACGACGAA 1641 UGACAGAGCCUGCUGAUAC 1642 AGGCGGCUCUGUACCAUUU1643 ADAM2 GAAACAUGCUGUGAUAUUG 1644 GCAGAUGUUUCCUUAUAUA 1645CAACAGAGAUGCCAUGAUA 1646 GAAAGGCGCUACAUUGAGA 1647 XPNPEP1GACCUGAGCUUCCCAACAA 1648 GCGACUGGCUCAACAAUUA 1649 GAGAUUGCGUGGCUAUUUA1650 GACAGCAACUGGACACUUA 1651 GZMA GGAAGAGACUCGUGCAAUG 1652GGAACCAUGUGCCAAGUUG 1653 GAAGUAACUCCUCAUUCAA 1654 GAACUCCUAUAGAUUUCUG1655 CMKLR1 CAUAGAAGCUUUACCAAGA 1656 GAAUGGAGGAUGAAGAUUA 1657GGUCAAUGCUCUAAGUGAA 1658 GAGAGGACUUCUAUGAAUG 1659 CLN3CAUCAUGCCUUCUGAAUAA 1660 CAACAGCUCAUCACGAUUU 1661 GCAACAACUUCUCUUAUGU1662 GGUCUUCGCUAGCAUCUCA 1663 CALCR GGACCUAGCUGUUGUAAAG 1664GAAAGACCAUGCAUUUAAA 1665 GCAGGAAGAUGUAUGCUUU 1666 GAAUAAACCAGUAUCGUUA1667 OXTR GGACCCAGAUAUCCAAAUA 1668 GCAAUACUAUCCUAACUGA 1669GAAUAUAGAUUAGCGUUUG 1670 GAUGAGGCAUGACUACUAA 1671 EDG4GCGAGUCUGUCCACUAUAC 1672 GAGAACGGCCACCCACUGA 1673 GAACGGCCACCCACUGAUG1674 GGUCAAUGCUGCUGUGUAC 1675 EDG5 UCCAGGAACACUAUAAUUA 1676GUGACCAUCUUCUCCAUCA 1677 CAUCCUCUGUUGCGCCAUU 1678 CCAACAAGGUCCAGGAACA1679 EDG7 ACACUGAUACUGUCGAUGA 1680 AAUAGGAGCAACACUGAUA 1681CAGCAGGAGUUACCUUGUU 1682 GGACACCCAUGAAGCUAAU 1683 PTCHGCACAGAACUCCACUCAAA 1684 GGACAGCAGUUCAUUGUUA 1685 GAGAAGAGGCUAUGUUUAA1686 GGACAAACUUCGACCCUUU 1687 SMO UCGCUACCCUGCUGUUAUU 1688GCUACAAGAACUACCGAUA 1689 CAAGAAAGCUUCCUUCAAC 1690 GAGAAGAAAUACAGUCAAU1691 CASP3 CAAUAUAUCUGAAGAGCUA 1692 GAACUGGACUGUGGCAUUG 1693GUGAGAAGAUGGUAUAUUU 1694 GAGGGUACUUUAAGACAUA 1695 CASP6CAUGAGGUGUCAACUGUUA 1696 GAAGUGAAAUGCUUUAAUG 1697 AAAUAUGGCUCCUCCUUAG1698 GCAAUCACAUUUAUGCAUA 1699 CAACAUAACUGAGGUGGAU 1700CAUGGUACAUUCAAGAUUU 1701 CASP7 GAACUCUACUUCAGUCAAU 1704GGGCAAAUGCAUCAUAAUA 1703 CAACAGAGGGAGUUUAAUA 1704 GAACAAAGCCACUGACUGA1705 CASP8 GAAGUGAACUAUGAAGUAA 1706 CAACAAGGAUGACAAGAAA 1707GGACAAAGUUUACCAAAUG 1708 GAGGGUCGAUCAUCUAUUA 1709 GAAUAUAGAGGGCUUAUGA1710 CAACGACUAUGAAGAAUUC 1711 GAAGUGAGCAGAUCAGAAU 1712GAGGAAAUCUCCAAAUGCA 1713 CASP9 CCAGGCAGCUGAUCAUAGA 1714UCUCAGGUGUUGCCAAAUA 1715 GAACAGCUGUAAUCUAUGA 1716 CCACUGGUCUGUAGGGAUU1717 DVL1 UCGUAAAGCUGUUGAUAUC 1718 GAGGAGAUCUUUGAUGACA 1719GUAAAGCUGUUGAUAUCGA 1720 GAUCGUAAAGCUGUUGAUA 1721 DVL2AGACGAAGGUGAUUUACCA 1722 UGUGAGAGGUACCUAGUCA 1723 GAAGAAAUUUCAGAUGACA1724 UAAUAGGCAUUUCCUCUUU 1725 PTEN GUGAAGAUCUUGACCAAUG 1726GAUCAGCAUACACAAAUUA 1727 GAAUGAACCUUCUGCAACA 1728 GGCGCUAUGUGUAUUAUUA1729 PDK1 GUACAAAGCUGGUAUAUCC 1730 GAAAGACUCCCAGUGUAUA 1731GGAAGUCCAUCUCAUCGAA 1732 CCAAAGACAUGACGACGUU 1733 PDK2GUAAAGAGGAGACUGAAUG 1734 GGUCUGUGAUGGUCCCUAA 1735 CAAAGAUGCCUACGACAUG1736 GGGCGAUGCCUGAGGGUUA 1737 PPP2CA UCACACAAGUUUAUGGUUU 1738CAACAGCCGUGACCACUUU 1739 UAACCAAGCUGCAAUCAUG 1740 GAACUUGACGAUACUCUAA1741 CTNNA1 GAAGAGAGGUCGUUCUAAG 1742 AAGCAGAUGUGCAUGAUUA 1743UCUAAUAACUGCAGUGUUU 1744 GUAAAGGGCCCUCUAAUAA 1745 CTNNA2GAAAGAAUAUGCCCAAGUU 1746 GAAGAAGAAUGCCACAAUG 1747 GCAGGAAGAUUAUGAUGUG1748 AAAGAAAGCCCAUGUACUA 1749 HSPCA GGGAAAGAGCUGCAUAUUA 1750GCUUAGAACUCUUUACUGA 1751 UAUAAGAGCUUGACCAAUG 1752 GCAGAUAUCUCUAUGAUUG1753 DCTN2 CAACUCAUGUCCAAUACUG 1754 GGAAUGAGCCAGAUGUUUA 1755GGAGACAGCUGUACGUUGU 1756 UCCAAGAGCUGACAACUGA 1757 CD2GUAAGGAGAAGCAAUAUAA 1758 AAGAUGAGCUUUCCAUGUA 1759 GGACAUCUAUCUCAUCAUU1760 GACAAGAGCCCACAGAGUA 1761 BAD GUACUUCCCUCAGGCCUAU 1762GCUGUGCCUUGACUACGUA 1763 GUACUUCCCUCAGGCCUAU 1764 GGUCAGGUGCCUCGAGAUC1765 SMAC CAGCGUAACUUCAUUCUUC 1766 UAACUUCAUUCUUCAGGUA 1767CAGCUGCUCUUACCCAUUU 1768 GAUUGAAGCUAUUACUGAA 1769 UAGAAGAGCUCCGUCAGAA1770 CCACAUAUGCGUUGAUUGA 1771 GCGCAGGGCUCUCUACCUA 1772 MAP3K5GAACAGCCUUCAAAUCAAA 1773 GAUGUUCUCUACUAUGUUA 1774 GCAAAUACUGGAAGGAUUA1775 CAGGAAAGCUCGUAAUUUA 1776 PVR CCACACGGCUGACCUCAUA 1777CAGCAGAAUUCCUCUUAUA 1778 GCAGAAUUCCUCUUAUAAA 1779 GAUCGGGAUUUAUUUCUAU1780 ERBB2 UGUGGGAGCUGAUGACUUU 1781 UCACAGAGAUCUUGAAAGG 1782UGGAAGAGAUCACAGGUUA 1783 GCUCAUCGCUCACAACCAA 1784 SOS1GAGCACCACUUCUAUGAUU 1785 CAAAGAAGCUGUUCAAUAU 1786 UGAAAGCCCUCCCUUAUUA1787 GAAAUAGCAUGGAGAAGGA 1788 BRCA1 CCAUACAGCUUCAUAAAUA 1789GAAGAGAACUUAUCUAGUG 1790 GAAGUGGGCUCCAGUAUUA 1791 GCAAGAUGCUGAUUCAUUA1792 GAAGUGGGCUCCAGUAUUA 1793 GAACGGACACUGAAAUAUU 1794GCAGAUAGUUCUACCAGUA 1795 CDKN1A GAACAAGGAGUCAGACAUU 1796AAACUAGGCGGUUGAAUGA 1797 GAUGGAACUUCGACUUUGU 1798 GUAAACAGAUGGCACUUUG1799 CDKN1B GGAAUGGACAUCCUGUAUA 1800 GGAGAAAGAUGUCAAACGU 1801GAAUGGACAUCCUGUAUAA 1802 GUAAACAGCUCGAAUUAAG 1803 SLC2A4CAGAUAGGCUCCGAAGAUG 1804 AGACUCAGCUCCAGAAUAC 1805 GAUCGGUUCUUUCAUCUUC1806 CAGGAUCGGUUCUUUCAUC 1807 NOS2A CCAGAUAAGUGACAUAAGU 1808UAAGUGACCUGCUUUGUAA 1809 GAAGAGAGAUUCCAUUGAA 1810 UGAAAGAGCUCAACAACAA1811 FRAP1 GAGCAUGCCGUCAAUAAUA 1812 CAAGAGAACUCAUCAUAAG 1813CCAAAGUGCUGCAGUACUA 1814 UAAGAAAGCUAUCCAGAUU 1815 FKBP1AGAAACAAGCCCUUUAAGUU 1816 GAAUUACUCUCCAAGUUGA 1817 CAGCACAAGUGGUAGGUUA1818 GUUGAGGACUGAAUUACUC 1819 GAUGGCAGCUGUUUAAAUG 1820GAGUAUCCUUUCAGUGUUA 1821 TNFRSF1A CAAAGGAACCUACUUGUAC 1822GGAACCUACUUGUACAAUG 1823 GAACCUACUUGUACAAUGA 1824 GAGUGUGUCUCCUGUAGUA1825 IL1R1 GGACAAGAAUCAAUGGAUA 1826 GAACAAGCCUCCAGGAUUC 1827GGACUUGUGUGCCCUUAUA 1828 GAACACAAAGGCACUAUAA 1829 IRAK1CGAAGAAAGUGAUGAAUUU 1830 GCUCUUUGCCCAUCUCUUU 1831 UGAAAGACCUGGUGGAAGA1832 GCAAUUCAGUUUCUACAUC 1833 TRAF2 GAAGACAGAGUUAUUAAAC 1834UCACGAAGACAGAGUUAUU 1835 AGACAGAGUUAUUAAACCA 1836 CACGAAGACAGAGUUAUUA1837 GCUGAAGCCUGUCUGAUGU 1838 TRAF6 CAAAUGAUCUGAGGCAGUU 1839GUUCAUAGUUUGAGCGUUA 1840 GGAGAAACCUGUUGUGAUU 1841 GGACAAAGUUGCUGAAAUC1842 CAAAUGAUCUGAGGCAGUU 1843 GGAGAAACCUGUUGUGAUU 1844GGACAAAGUUGCUGAAAUC 1845 GUUCAUAGUUUGAGCGUUA 1846 TRADDUGAAGCACCUUGAUCUUUG 1847 GGGCAGCGCAUACCUGUUU 1848 GAGGAGCGCUGUUUGAGUU1849 GGACGAGGAGCGCUGUUUG 1850 GAGGAGCGCUGUUUGAGUU 1851GGAUGUCUCUCUCCUCUUU 1852 GCUCACUCCUUUCUACUAA 1853 UGAAGCACCUUGAUCUUUG1854 FADD GCACAGAUAUUUCCAUUUC 1855 GCAGUCCUCUUAUUCCUAA 1856GAACUCAAGCUGCGUUUAU 1857 GGACGAAUUGAGAUAAUAU 1858 IKBKEUAAGAACACUGCUCAUGAA 1859 GAGGCAUCCUGAAGCAUUA 1860 GAAGGCGGCUGCAGAACUG1861 GGAACAAGGAGAUCAUGUA 1862 IKBKG CUAUCGAGGUCGUUAAAUU 1863GAAUGCAGCUGGAAGAUCU 1864 GCGGCGAGCUGGACUGUUU 1865 CCAGACCGAUGUGUAUUUA1866 TNFRSF5 GGUCUCACCUCGCUAUGGU 1867 GAAAGCGAAUUCCUAGACA 1868GCACAAACAAGACUGAUGU 1869 GAAGGGCACCUCAGAAACA 1870 UCUCCCAACUUGUAUUAAA1871 RELA UCAAGUGUCUUCCAUCAUG 1872 UCAAGUGCCUUAAUAGUAG 1873GGAGUACCCUGAGGCUAUA 1874 GAUGAGAUCUUCCUACUGU 1875 ARHAGAGCUGGGCUAAGUAAAUA 1876 GACCAAAGAUGGAGUGAGA 1877 GGAAGAAACUGGUGAUUGU1878 GGCUGUAACUACUUUAUAA 1879 CDC42 GGACAUUUGUUUGCCAUUU 1880GGAGAACCAUAUACUCUUG 1881 GAACCAAUGCUUUCUCAUG 1882 GAAGACCUGUUAUGUAGAG1883 GAUCAAGAAUUGCAAUAUC 1884 GAAAAGGGGUGACCUAGUA 1885UGACAAACCUUAUGGAAAA 1886 ROCK1 GGAAUGAGCUUCAGAUGCA 1887GGACACAGCUGUAAGAUUG 1888 GACAAGAGAUUACAGAUAA 1889 GAAGAAACAUUCCCUAUUC1890 PAK1 GAGGGUGGUUUAUGAUUAA 1891 CAACAAAGAACAAUCACUA 1892GAAGAAAUAUACACGGUUU 1893 UACAUGAGCUUUACAGAUA 1894 PAK2GGUAGGAGAUGAAUUGUUU 1895 AGAAGGAACUGAUCAUUAA 1896 CUACAGACCUCCAAUAUCA1897 GAAACUGGCCAAACCGUUA 1898 PAK3 GAUUAUCGCUGCAAAGGAA 1899GAGAGUGCCUGCAAGCUUU 1900 GACAAGAGGUGGCCAUAAA 1901 UUAAAUCGCUGUCUUGAGA1902 PAK4 ACUAAGAGGUGAACAUGUA 1903 GAUCAUGAAUGUCCGAAGA 1904GAUGAGACCCUACUACUGA 1905 CAGCAAAGGUGCCAAAGAU 1906 PAK6UAAAGGCAGUUGUCCACUA 1907 GAAGGGACCUGCUUUCUUG 1908 GCAAAGACGUCCCUAAGAG1909 CCAAUGGGCUGGCUGCAAA 1910 PAK7 GAGCACGGCUUUAAUAAGU 1911CAAACUCCGUUAUGAUAUA 1912 GGAUAAAGUUGUCUGAUUU 1913 GGAAAUGCCUCCAUAAAUA1914 HDAC1 GGACAUCGCUGUGAAUUGG 1915 AGAAAGAAGUCACCGAAGA 1916GGACAAGGCCACCCAAUGA 1917 CCACAGCGAUGACUACAUU 1918 HDAC2GCUGUUAAAUUAUGGCUUA 1919 GCAAAGAAAGCUAGAAUUG 1920 CAUCAGAGAGUCUUAUAUA1921 CCAAUGAGUUGCCAUAUAA 1922 CREBBP GGCCAUAGCUUAAUUAAUC 1923GCACAGCCGUUUACCAUGA 1924 GGACAGCCCUUUAGUCAAG 1925 GAACUGAUUCCUGAAAUAA1926 BTRC CACAUAAACUCGUAUCUUA 1927 GAGAAGGCACUCAAGUUUA 1928AGACAUAGUUUACAGAGAA 1929 GCAGAGAGAUUUCAUAACU 1930 RIPK2GAACAUACCUGUAAAUCAU 1931 GGACAUCGACCUGUUAUUA 1932 UAAAUGAACUCCUACAUAG1933 GGAAUUAUCUCUGAACAUA 1934 VAV1 GCAGAAAUACAUCUACUAA 1935GCUAUGAGCUGUUCUUCAA 1936 CGACAAAGCUCUACUCAUC 1937 GCUCAACCCUGGAGACAUU1938 VAV2 GGACAAGACUCGCAGAUUU 1939 GCUGAGCGCUUUGCAAUAA 1940CAAGAAGUCUCACGGGAAA 1941 UCACAGAGGCCAAGAAAUU 1942 GRB2UGGAAGCCAUCGCCAAAUA 432 CAUCAGUGCAUGACGUUUA 1943 UGAAUGAGCUGGUGGAUUA1944 UGCCAAAACUUACCUAUAA 1945 PLCG1 GAGCUGCACUCCMUGAGA 1946GAAACCAAGCCAUUAAUGA 1947 CCAAGGAGCUACUGACAUU 1948 AGAGAAACAUGGCCCAAUA1949 ITGB1 CCACAGACAUUUACAUUAA 1950 GAAGGGAGUUUGCUAAAUU 1951GAACAGAUCUGAUGAAUGA 1952 CAAGAGAGCUGAAGACUAU 1953 ITGA4GCAUAUAUAUUCAGCAUUG 1954 CAACUUGACUGCAGUAUUG 1955 GAACUUAACUUUCCAUGUU1956 GACAAGACCUGUAGUAAUU 1957 STAT1 AGAAAGAGCUUGACAGUAA 1958GGAAGUAGUUCACAAAAUA 1959 UGAAGUAUCUGUAUCCAAA 1960 GAGCUUCACUCCCUUAGUU1961 KRAS2 UAAGGACUCUGAAGAUGUA 1962 GACAAAGUGUGUAAUUAUG 1963GCUCAGGACUUAGCAAGAA 1964 GAAACUGAAUACCUAAGAU 1965 GAAACUGAAUACCUAAGAU1966 UAAGGACUCUGAAGAUGUA 1967 GACAAAGUGUGUAAUUAUG 1968GCUCAGGACUUAGCAAGAA 1969 HRAS CCAUCCAGCUGAUCCAGAA 1970GAACCCUCCUGAUGAGAGU 1971 GAGGACAUCCACCAGUACA 1972 BRAFGAUUAGAGACCAAGGAUUU 2410 CCACUGAUGUGUGUUAAUU 1973 CAAUAGAACCUGUCAAUAU1974 GAAGACAGGAAUCGAAUGA 1975 ELK1 GAUGUGAGUAGAAGAGUUA 2411GGAAGAAUUUGUACCAUUU 1976 GAACGACCUUUCUUUCUUU 1977 GGAGUCAUCUCUUCCUAUA1978 RALGDS GGAGAAGCCUCACCUCUUG 1979 GCAGAAAGGACUCAAGAUU 1980GAGAACAACUACUCAUUGA 1981 GAACUUCUCGUCACUGUAU 1982 PRKCAGGAUUGUUCUUUCUUCAUA 1983 GAAGGGUUCUCGUAUGUCA 1984 GAAGAAGGAUGUGGUGAUU1985 GGACUGGGAUCGAACAACA 1986 MAP2K4 GGACAGAAGUGGAAAUAUU 1987UCAAAGAGGUGAACAUUAA 1988 GACCAAAUCUCAGUUGUUU 1989 GGAGAAUGGUGCUGUUUAA1990 MAP2K7 GAAGAGACCAAAGUAUAAU 1991 GAAGACCGGCCACGUCAUU 1992GGAAGAGACCAAAGUAUAA 1993 GCAUUGAGAUUGACCAGAA 1994 UGAGAGAACGAGAAAGUUG1995 GUGAAACCCUGUCUGCAUU 1996 GGAUCUCUCUCAACAACUA 1997ACAACUAGGUGAACACAUA 1998 MAPK8 UCACAGUCCUGAAACGAUA 1999GAUUGGAGAUUCUACAUUC 2000 GCUCAUGGAUGCAAAUCUU 2001 GAAGCUAAGCCGACCAUUU2002 MAPK9 AAAGAGAGCUUAUCGUGAA 2003 GAUGAUAGGUUAGAAAUAG 2004ACAAAGAAGUCAUGGAUUG 2005 GGAGCUGGAUCAUGAAAGA 2006 AIF1GAAAAGGGAUGAUGGGAUU 2007 CCUAGACGAUCCCAAAUAU 2008 GAGCCAAACCAGGGAUUUA2009 UGAAACGAAUGCUGGAGAA 2010 UCACUCACCCAGAGAAAUA 2011CCAAGAAAGCUAUCUCUGA 2012 AGACUCACCUAGAGCUAAA 2013 BBC3CCUGGAGGGUCCUGUACAA 2014 GAGCAAAUGAGCCAAACGU 2015 GGAGGGUCCUGUACAAUCU2016 GACUUUCUCUGCACCAUGU 2017 BCL2L1 CCAGGGAGCUUGAAAGUUU 2018AAAGUGCAGUUCAGUAAUA 2019 GAGAAUCACUAACCAGAGA 2020 GAGCCCAUCCCUAUUAUAA2021 BCL2L11 GAGACGAGUUUAACGCUUA 2022 AAAGCAACCUUCUGAUGUA 2023CCGAGAAGGUAGACAAUUG 2024 GCAAAGCAACCUUCUGAUG 2025 AGACAGAGCCACAAGGUAA2026 GCAAGGAGGUUAGAGAAAU 2027 CAAGGAGGUUAGAGAAAUA 2028UCUUACGACUGUUACGUUA 2029 BID GAAGACAUCAUCCGGAAUA 2030CAACAGCGUUCCUAGAGAA 2031 GAAAUGGGAUGGACUGAAC 2032 ACGAUGAGCUGCAGACUGA2033 BIRC2 GAAAGAAGCCUGCAUAUAA 2034 GAAAUUGACUCUACAUUGU 2035ACAAAUAGCACUUAGGUUA 2036 GAAUACACCUGUGGUUAAA 2037 BIRC3GGAGAUGCCUGCCAUUAAA 2038 UCAAUGAUCUUGUGUUAGA 2039 GAAAGAACAUGUAAAGUGU2040 GAAGAAAGAACAUGUAAAG 2041 BIRC4 GUAGAUAGAUGGCAAUAUG 2042GAGGAGGGCUAACUGAUUG 2043 GAGGAACCCUGCCAUGUAU 2044 GCACGGAUCUUUACUUUUG2045 BIRC5 GGCGUAAGAUGAUGGAUUU 2046 GCAAAGGAAACCAACAAUA 2047GCACAAAGCCAUUCUAAGU 2048 CAAAGGAAACCAACAAUAA 2049 BRCA1CCAUACAGCUUCAUAAAUA 2050 GAAGAGAACUUAUCUAGUG 2051 GAAGUGGGCUCCAGUAUUA2052 GCAAGAUGCUGAUUCAUUA 2053 CCAUACAGCUUCAUAAAUA 2054 CARD4GAAAGUUAAUGUCAAGGAA 2055 GAGCAACACUGGCAUAACA 2056 UAACAGAGAUUUGCCUAAA2057 GCGAAGAGCUGACCAAAUA 2058 CASP10 CAAAGGGUUUCUCUGUUUA 2059GAAAUGACCUCCCUAAGUU 2060 GAAGGCAGCUGGUAUAUUC 2061 GACAUGAUCUUCCUUCUGA2062 GCACUCUUCUGUUCCCUUA 2063 CASP2 GUAUUAAACUCUCCUUUGA 2064GCAAGGAGAUGUCUGAAUA 2065 CAACUUCCCUGAUCUUUAA 2066 GCUCAAAGAUGUAAUGUAG2067 CDKN1A GAACAAGGAGUCAGACAUU 2068 AAACUAGGCGGUUGAAUGA 2069GAUGGAACUUCGACUUUGU 2070 GUAAACAGAUGGCACUUUG 2071 CFLARGAUGUGUCCUCAUUAAUUU 2072 GAAGAGAGAUACAAGAUGA 2073 GAGCAUACCUGAAGAGAGA2074 GCUAUGAAGUCCAGAAAUU 2075 CLK2 GUGAAUAUGUGAAAUAGUG 2076AAAGCAUGCUAGAGUAUGA 2077 UUAAGAAUGUGGAGAAGUA 2078 GAUAACAAGCUGACACAUA2079 CLSPN GGACGUAAUUGAUGAAGUA 2080 GCAGAUGGGUUCUUAAAUG 2081CAAAUGAGGUUGAGGAAAU 2082 GGAAAUACCUGGAGGAUGA 2083 CSNK2A1GAUCCACGUUUCAAUGAUA 2084 GCAUUUAGGUGGAGACUUC 2085 GAUGUACGAUUAUAGUUUG2086 UGAAUUAGAUCCACGUUUC 2087 CTNNB1 GCACAAGAAUGGAUCACAA 2088GCUGAAACAUGCAGUUGUA 2089 GUACGUACCAUGCAGAAUA 2090 GAACUUGCAUUGUGAUUGG2091 CXCR4 GAAGCAUGACGGACAAGUA 2092 GAACAUUCCAGAGCGUGUA 2093GUUCUUAGUUGCUGUAUGU 2094 CAUCAUGGUUGGCCUUAUC 2095 CXCR6GGAACAAACUGGCAAAGCA 2096 GAUCAGAGCAGCAGUGAAA 2097 GGGCAAAACUGAAUUAUAA2098 GAUCUCAGGUUCUCCUUGA 2099 DAXX CUACAGAUCUCCAAUGAAA 2100GCUACAAGCUGGAGAAUGA 2101 GGAAACAGCUAUGUGGAAA 2102 GGAGUUGGAUCUCUCAGAA2103 GAS41 GUAGUAAGCUAAACUGAAA 2104 GACAAUAUGUUCAAGAGAA 2105GACAACAUCUCGUCAGCUA 2106 UAUAUGAUGUGUCCAGUAA 2107 GTSE1CAAAGAAGCUCACUUACUG 2108 GAACAGCCCUAAAGUGGUU 2109 GAACAUGGAUGACCCUAAG2110 GGGCAAAGCUAAAUCAAGU 2111 HDAC3 GGAAAGCGAUGUGGAGAUU 2112CCAAGACCGUGGCCUAUUU 2113 AAAGCGAUGUGGAGAUUUA 2114 GUGAGGAGCUUCCCUAUAG2115 HDAC5 GAAUUCCUCUUGUCGAAGU 2116 GUUAUUAGCACCUUUAAGA 2117GGAGGGAGGCCAUGACUUG 2118 CAGGAGAGCUCAAGAAUGG 2119 GGAUAUGGAUUUCAGUUAA2120 GGAAGUCGGUGCCUUGGUU 2121 GGAAGGAGAGGACUGGUUU 2122 HECGCAGAUACUUGCACGGUUU 2123 GAGUAGAACUAGAAUGUGA 2124 GCGAAUAAAUCAUGAAAGA2125 GAAGAUGGAAUUAUGCAUA 2126 HIST1H2 GGCAAUGCGUCUCGCGAUA 2127 AAGAUCCGCAAUGAUGAGGAA 2128 GCAAUGCGUCUCGCGAUAA 2129 GAGGAACUCAAUAAGCUUU2130 LMNB1 AAUAGAAGCUGUGCAAUUA 2131 CAACUGACCUCAUCUGGAA 2132GAAGGAAUCUGAUCUUAAU 2133 GGGAAGGGUUUCUCUAUUA 2134 LMNB2GGAGGUUCAUUGAGAAUUG 2134 GGCAAUAGCUCACCGUUUA 2135 CAAAUACGCUUAGCUGUGU2136 GGAGAUCGCCUACAAGUUC 2137 MYB GCAGAAACACUCCAAUUUA 2138GUAAAUACGUGAAUGCAUU 2139 GCACUGAACUUUUGAGAUA 2140 GAAGAACAGUCAUUUGAUG2141 MYT1 GAGGUGAGCUGUUAAAUCA 2142 GCAGGGUGAUUUCCUAAUA 2143GGGAGAAGAUAUUUAAUUG 2144 CAACUUCUCUCCUGAACUU 2145 NFKBIBGGACACGGCACUGCACUUG 2146 GCACUUGGCUGUGAUUCAU 2148 GAGACGAGGGCGAUGAAUA2149 CAUGAACCCUUCCUGGAUU 2150 NFKBIA GAACAUGGACUUGUAUAUU 2151GAUGUGGGGUGAAAAGUUA 2152 GGACGAGAAAGAUCAUUGA 2153 AGGACGAGCUGCCCUAUGA2154 NFKBIE GAAGGGAAGUUUCAGUAAC 2155 GGAAGGGAAGUUUCAGUAA 2156GAAACUGCUGCUGUGUAC 2157 GAACCAACCACUCAUGGAA 2158 NUMA1GGGAACAGUUUGAAUAUAA 2159 GCAGUAGCCUGAAGCAGAA 2160 CGAGAAGGAUGCACAGAUA2161 GCAAGAGGCUGAGAGGAAA 2162 NUP153 GAAGACAAAUGAAAGCUAA 2163GAUAAAGACUGCUGUUAGA 2164 GAGGAGAGCUCUAAUAUUA 2165 GAGGAAGCCUGAUUAAAGA2166 OPA1 GAAAGAGCAUGAUGACAUA 2167 GAGGAGAGCUCUAUUAUGU 2168GAAACUGAAUGGAAGAAUA 2169 AAAGAAGGCUGUACCGUUA 2170 PARVACUACAUGUCUUUGCUCUUA 2171 GCUAAGUCCUGUAAGAAUA 2172 CAAAGGCAAUGUACUGUUU2173 GAACAAUGGUGGAUCCAAA 2174 PIK3CG AAGUUCAGCUUCUCUAUUA 2175GAAGAAAUCUCUGAUGGAU 2176 GAACACCUUUACUCUAUAA 2177 GCAUGGAGCUGGAGAACUA2178 PRKDC AUGAAAGCUCUAAAGAUG 2179 AAAGGAGGUUCUAAACUA 2180GAAGAAGCUCAUUUGAUU 2181 GCAAAGAGGUGGCAGUUAA 2182 RASA1GGAAGAAGAUCCACAUGAA 2183 GAACAUACUUUCAGAGCUU 2184 GAACAAUCUUUGCUGUAUA2185 UAACAGAACUGCUUCAACA 2186 SLC9A1 GAAGAGAUCCACACACAGU 2187UCAAUGAGCUGCUGCACAU 2188 GAAGAUAGGUUUCCAUGUG 2189 GAAUUACCCUUCCUCAUCU2190 TEGT CUACAGAGCUUCAGUGUGA 2191 GAACAUAUUUGAUCGAAAG 2192GAGCAAACCUAGAUAAGGA 2193 GCAUUGAUCUCUUCUUAGA 2194 TERTGGAAGACAGUGGUGAACUU 2195 GCAAAGCAUUGGAAUCAGA 2196 GAGCUGACGUGGAAGAUGA2197 GAACGGGCCUGGAACCAUA 2198 TNFRSF6 GAUACUAACUGCUCUCAGA 2199GAAAGAAUGGUGUCAAUGA 2200 UCAAUAAUGUCCCAUGUAA 2201 UCAUGAAUCUCCAACCUUA2202 GAUGUUGACUUGAGUAAAU 2203 TOP1 AAAGGAAAUGACUAAUGA 2204AAGAAGGCUGUUCAGAGA 2205 GAAGUAGCUACGUUCUUU 2206 GACAUAAGUGGAAAGAAG 2207TOP2A GAAAGAGUCCAUCAGAUUU 2208 CAAACUACAUUGGCAUUUA 2209AAACAGACAUGGAUGGAUA 2210 CGAAAGGAAUGGUUAACUA 2211 TOP3ACCAGAAAUCUUCCACAGAA 2212 GAAACUAUCUGGAUGUGUA 2213 CCACAAAGAUGGUAUCGUA2214 GGAAAUGGCUGUGGUAACA 2215 TOP3B GAGACAAGAUGAAGACUGU 2216GCACAUGGGCUGCGUCUUU 2217 CCAGUGCGCUUCAAGAUGA 2218 GAACAUCUGCUUUGAGGUU2219 WEE1 GGUAUUGCCUUGUGAAUUU 2220 GCAGAACAAUUACGAAUAG 2221GUACAUAGCUGUUUGAAAU 2222 GCUGUAAACUUGUAGCAUU 2223

In addition, to identifying functional siRNA against gene families orpathways, it is possible to design duplexes against genes known to beinvolved in specific diseases. For example when dealing with humandisorders associated with allergies, it will be beneficial to developsiRNA against a number of genes including but not limited to: theinterleukin 4 receptor gene (SEQ. ID NO. 2224: UAGAGGUGCUCAUUCAUUU, SEQ.ID NO. 2225: GGUAUAAGCCUUUCCAAGA, SEQ. ID NO. 2412: ACACACAGCUGGAAGAAAU,SEQ. ID NO. 2226: UAACAGAGCUUCCUUAGGU), the Beta-arrestin-2 (SEQ. ID NO.2227: GGAUGAAGGAUGACGACUA, SEQ. ID NO. 2228: ACACCAACCUCAUUGAAUU, SEQ.ID NO. 2229: CGAACAAGAUGACCAGGUA, SEQ. ID NO. 2230:GAUGAAGGAUGACGACUAU,), the interferon-gamma receptor 1 gene (SEQ. ID NO.2231: CAGCAUGGCUCUCCUCUUU, SEQ. ID NO. 2232: GUAAAGAACUAUGGUGUUA, SEQ.ID NO. 2233: GAAACUACCUGUUACAUUA, SEQ. ID NO. 2234:GAAGUGAGAUCCAGUAUAA), the matrix metalloproteinase MMP-9 (SEQ. ID NO.2235: GGAACCAGCUGUAUUUGUU, SEQ. ID NO. 2236: GUUGGAGUGUUUCUAAUAA, SEQ.ID NO. 2237: GCGCUGGGCUUAGAUCAUU, SEQ. ID NO. 2238:GGAGCCAGUUUGCCGGAUA), the Slclla1 (Nrampl) gene (SEQ. ID NO. 2239:CCAAUGGCCUGCUGAACAA, SEQ. ID NO. 2240: GGGCCUGGCUUCCUCAUGA, SEQ. ID NO.2241: GGGCAGAGCUCCACCAUGA, SEQ. ID NO. 2242: GCACGGCCAUUGCAUUCAA),SPINK5 (SEQ. ID NO. 2243: CCAACUGCCUGUUCAAUAA, SEQ. ID NO. 2244:GGAUACAUGUGAUGAGUUU, SEQ. ID NO. 2245: GGACGAAUGUGCUGAGUAU, SEQ. ID NO.2246: GAGC1JUGUCUUAUUUGCUA,), the CYP1A2 gene (SEQ. ID NO. 2247:GAAAUGCUGUGUCUUCGUA, SEQ. ID NO. 2248: GGACAGCACUUCCCUGAGA, SEQ. ID NO.2249: GAAGACACCACCAUUCUGA, SEQ. ID NO. 2250: GGCCAGAGCUUGACCUUCA),thymosin-beta4Y (SEQ. ID NO. 2251: GGACAGGCCUGCGUUGUUU, SEQ. ID NO.2252: GGAAAGAGGAAGCUCAUGA, SEQ. ID NO. 2253: GCAAACACGUUGGAUGAGU, SEQ.ID NO. 2254: GGACUAUGCUGCCCUUUUG, activin A receptor IB (SEQ. ID NO.2255: ACAAGACGCUCCAGGAUCU, SEQ. ID NO. 2413: GCAACAGGAUCGACUUGAG, SEQ.ID NO. 2414: GAAGCUGCGUCCCAACAUC, SEQ. ID NO. 2256: GCAUAGGCCUGUAAUCGUA,SEQ. ID NO. 2257: UCAGAGAGUUCGAGACAAA, SEQ. ID NO. 2258:UGCGAAAGGUUGUAUGUGA, SEQ. ID NO. 2259: GCAACAGGAUCGACUUGAG, SEQ. ID NO.2260: GAAUAGCGUUGUGUGUUAU, SEQ. ID NO. 2261: UGAAUAGCGUUGUGUGUUA, SEQ.ID NO. 2262: GGGAUCAGUUUGUUGAAUA, SEQ. ID NO. 2263:GAGCCUGAAUCAUCGUUUA,), ADAM33 (SEQ. ID NO. 2264: GGAAGUACCUGGAACUGUA,SEQ. ID NO. 2265: GGACAGAGGGAACCAUUUA, SEQ. ID NO. 2266:GGUGAGAGGUAGCUCCUAA, SEQ. ID NO. 2267: AAAGACAGGUGGCCACUGA), the TAP 1gene (SEQ. ID NO. 2268: GAAAGAUGAUCAGCUAUUU, SEQ. ID NO. 2269:CAACAGAACCAGACAGGUA, SEQ. ID NO. 2270: UGAGAAAUGUUCAGAAUGU, SEQ. ID NO.2271: UACCUUCACUCGAAACUUA, COX-2 (SEQ. ID NO. 2272: GAACGAAAGUAAAGAUGUU,SEQ. ID NO. 2273: GGACUUAUGGGUAAUGUUA, SEQ. ID NO. 2274:UGAAAGGACUUAUGGGUAA, SEQ. ID NO. 2275: GAUCAGAGUUCACUUUCUU), ADPRT (SEQ.ID NO. 2276: GGAAAGAUGUUAAGCAUUU, SEQ. ID NO. 2277: CAUGGGAGCUCUUGAAAUA,SEQ. ID NO. 2278: GAACAAGGAUGAAGUGAAG, SEQ. ID NO. 2279:UGAAGAAGCUCACAGUAAA,), HDC (SEQ. ID NO. 2280: CAGCAGACCUUCAGUGUGA, SEQ.ID NO. 2281: GGAGAGAGAUGGUGGAUUA, SEQ. ID NO. 2282: GUACAGAGCUGGAGAUGAA,SEQ. ID NO. 2283: GAACGUCCCUUCAGUCUGU), HnmT (SEQ. ID NO. 2284:CAAAUUCUCUCCAAAGUUC, SEQ. ID NO. 2285: GGAUAUAUCUGACUGCUUU, SEQ. ID NO.2286: GAGCAGAGCUUGGGAAAGA, SEQ. ID NO. 2287: GAUAUGAGAUGUAGCAAAU),GATA-3 (SEQ. ID NO. 2288: GAACUGCUUUCUUUCGUUU, SEQ. ID NO. 2289:GCAGUAUCAUGAAGCCUAA, SEQ. ID NO. 2290: GAAACUAGGUCUGAUAUUC, SEQ. ID NO.2291: GUACAGCUCCGGACUCUUC), Gab2 (SEQ. ID NO. 2292: GCACAACCAUUCUGAAGUU,SEQ. ID NO. 2293: GGACUUAGAUGCCCAGAUG, SEQ. ID NO. 2294:GAAGGUGGAUUCUAGGAAA, SEQ. ID NO. 2295: GGACUAGCCCUGCUGUUUA), and STAT6(SEQ. ID NO. 2296: GAUAGAAACUCCUGCUAAU, SEQ. ID NO. 2297:GGACAUUUAUUCCCAGCUA, SEQ. ID NO. 2298: GGACAGAGCUACAGACCUA, SEQ. ID NO.2299: GGAUGGCUCUCCACAGAUA).

In addition, rationally designed siRNA or siRNA pools can be directedagainst genes involved in anemia, hemophila or hypercholesterolemia.Such genes would include, but are not be limited to: APOA5 (SEQ. ID NO.2300: GAAAGACAGCCUUGAGCAA, SEQ. ID NO. 2301: GGACAGGGAGGCCACCAAA, SEQ.ID NO. 2302: GGACGAGGCUUGGGCUUUG, SEQ. ID NO. 2303:AGCAAGACCUCAACAAUAU), HMG-CoA reductase (SEQ. ID NO. 2304:GAAUGAAGCUUUGCCCUUU, SEQ. ID NO. 2305: GAACACAGUUUAGUGCUUU, SEQ. ID NO.2306: UAUCAGAGCUCUUAAUGUU, SEQ. ID NO. 2307: UGAAGAAUGUCUACAGAUA), NOS3(SEQ. ID NO. 2308: UGAAGCACCUGGAGAAUGA, SEQ. ID NO. 2309:CGGAACAGCACAAGAGUUA, SEQ. ID NO. 2310: GGAAGAAGACCUUUAAAGA, SEQ. ID NO.2415: GCACAAGAGUUAUAAGAUC), ARH (SEQ. ID NO. 2416: CGAUACAGCUUGGCACUUU,SEQ. ID NO. 2311: GAGAAGCGCUGCCCUGUGA, SEQ. ID NO. 2312:GAAUCAUGCUGUUCUCUUU, SEQ. ID NO. 2313: GGAGUAACCGGACACCUUA), CYP7A1(SEQ. ID NO. 2314: UAAGGUGACUCGAGUGUUU, SEQ. ID NO. 2315:AAACGACACUUUCAUCAAA, SEQ. ID NO. 2316: GGACUCAAGUUAAAGUAUU, SEQ. ID NO.2317: GUAAUGGACUCAAGUUAAA), FANCA (SEQ. ID NO. 2318:GGACAUCACUGCCCACUUC, SEQ. ID NO. 2319: AGAGGAAGAUGUUCACUUA, SEQ. ID NO.2320: GAUCGUGGCUCUUCAGGAA, SEQ. ID NO. 2321: GGACAGAGGCAGAUAAGAA), FANCG(SEQ. ID NO. 2322: GCACUAAGCAGCCUUCAUG, SEQ. ID NO. 2323:GCAAGCAGGUGCCUACAGA, SEQ. ID NO. 2324: GGAAUUAGAUGCUCCAUUG, SEQ. ID NO.2325: GGACAUCUCUGCCAAAGUC), ALAS (SEQ. ID NO. 2326: CAAUAUGCCUGGAAACUAU,SEQ. ID NO. 2327: GGUUAAGACUCACCAGUUC, SEQ. ID NO. 2328:CAACAGGACUUUAGGUUCA, SEQ. ID NO. 2329: GCAUAAGAUUGACAUCAUC), PIGA (SEQ.ID NO. 2330: GAAAGAGGGCAUAAGGUUA, SEQ. ID NO. 2331: GGACUGAUCUUUAAACUAU,SEQ. ID NO. 2332: UCAAAUGGCUUACUUCAUC, SEQ. ID NO. 2333:UCUAAGAACUGAUGUCUAA), and factor VIII (SEQ. ID NO. 2334:GCAAAUAGAUCUCCAUUAC, SEQ. ID NO. 2335: CCAGAUAUGUCGUUCUUUA, SEQ. ID NO.2336: GAAAGGCUGUGCUCUCAAA, SEQ. ID NO. 2337: GGAGAAACCUGCAUGAAAG, SEQ.ID NO. 2338: CUUGAAGCCUCCUGAAUUA, SEQ. ID NO. 2339: GAGGAAGCAUCCAAAGAUU,SEQ. ID NO. 2340: GAUAGGAGAUACAAACUUU).

Furthermore, rationally designed siRNA or siRNA pools can be directedagainst genes involved in disorders of the brain and nervous system.Such genes would include, but are not be limited to: APBB1 (SEQ. ID NO.2341: CUACGUAGCUCGUGAUAAG, SEQ. ID NO. 2342: GCAGAGAUGUCCACAGGUU, SEQ.ID NO. 2343: CAUGAGAUCUGCUCUAAGA, SEQ. ID NO. 2344:GGGCACCUCUGCUGUAUUG), BACE1 (SEQ. ID NO. 2345: CCACAGAGCAAGUGAUUUA, SEQ.ID NO. 2346: GCAGAAAGGAGAUCAUUUA, SEQ. ID NO. 2347: GUAGCAAGAUCUUUACAUA,SEQ. ID NO. 2348: UGUCAGAGCUUGAUUAGAA), PSEN1 (SEQ. ID NO. 2349:GAGCUGACAUUGAAAUAUG, SEQ. ID NO. 2350: GUACAGCUAUUUCUCAUCA, SEQ. ID NO.2351: GAGGUUAGGUGAAGUGGUU, SEQ. ID NO. 2352: GAAAGGGAGUCACAAGACA, SEQ.ID NO. 2353: GAACUGGAGUGGAGUAGGA, SEQ. ID NO. 2354: CAGCAGGCAUAUCUCAUUA,SEQ. ID NO. 2355: UCAAGUACCUCCCUGAAUG), PSEN2 (SEQ. ID NO. 2356:GCUGGGAAGUGGCUUAAUA, SEQ. ID NO. 2357: CAUAUUCCCUGCCCUGAUA, SEQ. ID NO.2358: GGGAAGUGCUCAAGACCUA, SEQ. ID NO. 2359: CAUAGAAAGUGACGUGUUA), MASS1 (SEQ. ID NO. 2360: GGAAGGAGCUGUUAUGAGA, SEQ. ID NO. 2361:GAAAGGAGAAGCUAAAUUA, SEQ. ID NO. 2362: GGAGGAAGGUCAAGAUUUA, SEQ. ID NO.2363: GGAAAUAGCUGAGAUAAUG,), ARX (SEQ. ID NO. 2364: CCAGACGCCUGAUAUUGAA,SEQ. ID NO. 2365: CAGCACCACUCAAGACCAA, SEQ. ID NO. 2366:CGCCUGAUAUUGAAGUAAA, SEQ. ID NO. 2367: CAACAUCCACUCUCUCUUG) and NNMT(SEQ. ID NO. 2368: GGGCAGUGCUCCAGUGGUA, SEQ. ID NO. 2369:GAAAGAGGCUGGCUACACA, SEQ. ID NO. 2370: GUACAGAAGUGAGACAUAA, SEQ. ID NO.2371: GAGGUGAUCUCGCAAAGUU).

In addition, rationally designed siRNA or siRNA pools can be directedagainst genes involved in hypertension and related disorders. Such geneswould include, but are not be limited to: angiotensin II type 1 receptor(SEQ. ID NO. 2372: CAAGAAGCCUGCACCAUGU, SEQ. ID NO. 2373:GCACUUCACUACCAAAUGA, SEQ. ID NO. 2374: GCACUGGUCCCAAGUAGUA, SEQ. ID NO.2375: CCAAAGGGCAGUAAAGUUU, SEQ. ID NO. 2376: GCUCAGAGGAGGUGUAUUU, SEQ.ID NO. 2377: GCACUUCACUACCAAAUGA, SEQ. ID NO. 2378: AAAGGGCAGUAAAGUUU),AGTR2 (SEQ. ID NO. 2379: GAACAUCUCUGGCAACAAU, SEQ. ID NO. 2380:GGUGAUAUAUCUCAAAUUG, SEQ. ID NO. 2381: GCAAGCAUCUUAUAUAGUU, SEQ. ID NO.2382: GAACCAGUCUUUCAACUCA),and other related targets.

Example XIII Validation of Multigene Knockout Using Rab5 and Eps

Two or more genes having similar, overlapping functions often leads togenetic redundancy. Mutations that knockout only one of, e.g., a pair ofsuch genes (also referred to as homologs) results in little or nophenotype due to the fact that the remaining intact gene is capable offulfilling the role of the disrupted counterpart. To fully understandthe function of such genes in cellular physiology, it is often necessaryto knockout or knockdown both homologs simultaneously. Unfortunately,concomitant knockdown of two or more genes is frequently difficult toachieve in higher organisms (e.g., mice) thus it is necessary tointroduce new technologies dissect gene function. One such approach toknocking down multiple genes simultaneously is by using siRNA. Forexample, FIG. 11 showed that rationally designed siRNA directed againsta number of genes involved in the clathrin-mediated endocytosis pathwayresulted in significant levels of protein reduction (e.g., >80%). Todetermine the effects of gene knockdown on clathrin-related endocytosis,internalization assays were performed using epidermal growth factor andtransferrin. Specifically, mouse receptor-grade EGF (CollaborativeResearch Inc.) and iron-saturated human transferrin (Sigma) wereiodinated as described previously (Jiang, X., Huang, F., Marusyk, A. &Sorkin, A. (2003) Mol Biol Cell 14, 858-70). HeLa cells grown in 12-welldishes were incubated with ¹²⁵I-EGF (1 μg/ml) or ¹²⁵I-transferrin (1μg/ml) in binding medium (DMEM, 0.1% bovine serum albumin) at 37° C.,and the ratio of internalized and surface radioactivity was determinedduring 5-min time course to calculate specific internalization rateconstant k_(e) as described previously (Jiang, X et al.). Themeasurements of the uptakes of radiolabeled transferrin and EGF wereperformed using short time-course assays to avoid influence of therecycling on the uptake kinetics, and using low ligand concentration toavoid saturation of the clathrin-dependent pathway (for EGF Lund, K. A.,Opresko, L. K., Strarbuck, C., Walsh, B. J. & Wiley, H. S. (1990) J.Biol. Chem. 265, 15713-13723).

The effects of knocking down Rab5a, 5b, 5c, Eps, or Eps 15R(individually) are shown in FIG. 22 and demonstrate that disruption ofsingle genes has little or no effect on EGF or Tfn internalization. Incontrast, simultaneous knock down of Rab5a, 5b, and 5c, or Eps and Eps15R, leads to a distinct phenotype (note: total concentration of siRNAin these experiments remained constant with that in experiments in whicha single siRNA was introduced, see FIG. 23). These experimentsdemonstrate the effectiveness of using rationally designed siRNA toknockdown multiple genes and validates the utility of these reagents tooverride genetic redundancy.

Example XIV Validation of Multigene Targeting Using G6PD, GAPDH, PLK,and UQC

Further demonstration of the ability to knock down expression ofmultiple genes using rationally designed siRNA was performed using poolsof siRNA directed against four separate genes. To achieve this, siRNAwere transfected into cells (total siRNA concentration of 100 nM) andassayed twenty-four hours later by B-DNA. Results shown in FIG. 24 showthat pools of rationally designed molecules are capable ofsimultaneously silencing four different genes.

Example XV Validation of Multigene Knockouts as Demonstrated by GeneExpression Profiling, a Prophetic Example

To further demonstrate the ability to concomitantly knockdown theexpression of multiple gene targets, single siRNA or siRNA poolsdirected against a collection of genes (e.g., 4, 8, 16, or 23 differenttargets) are simultaneously transfected into cells and cultured fortwenty-four hours. Subsequently, mRNA is harvested from treated (anduntreated) cells and labeled with one of two fluorescent probes dyes(e.g., a red fluorescent probe for the treated cells, a greenfluorescent probe for the control cells.). Equivalent amounts of labeledRNA from each sample is then mixed together and hybridized to sequencesthat have been linked to a solid support (e.g., a slide, “DNA CHIP”).Following hybridization, the slides are washed and analyzed to assesschanges in the levels of target genes induced by siRNA.

Example XVI Identifying Hyperfunctional siRNA

Identification of Hyperfunctional Bcl-2 siRNA

The ten rationally designed Bcl2 siRNA (identified in FIG. 13, 14) weretested to identify hyperpotent reagents. To accomplish this, each of theten Bcl-2 siRNA were individually transfected into cells at a 300 pM(0.3 nM) concentrations. Twenty-four hours later, transcript levels wereassessed by B-DNA assays and compared with relevant controls. As shownin FIG. 25, while the majority of Bcl-2 siRNA failed to inducefunctional levels of silencing at this concentration, siRNA 1 and 8induced >80% silencing, and siRNA 6 exhibited greater than 90% silencingat this subnanomolar concentration.

By way of prophetic examples, similar assays could be performed with anyof the groups of rationally designed genes described in Example VII orExample VIII. Thus for instance, rationally designed siRNA sequencesdirected against

PDGFA

(SEQ. ID NO. 2383: GGUAAGAUAUUGUGCUUUA,

SEQ. ID NO. 2384: CCGCAAAUAUGCAGAAUUA,

SEQ. ID NO. 2385: GGAUGUACAUGGCGUGUUA,

SEQ. ID NO. 2386: GGUGAAGUUUGUAUGUUUA), or

PDGFB

(SEQ. ID NO. 2387: GCUCCGCGCUUUCCGAUUU,

SEQ. ID NO. 2388: GAGCAGGAAUGGUGAGAUG,

SEQ. ID NO. 2389: GAACUUGGGAUAAGAGUGU,

SEQ. ID NO. 2390: CCGAGGAGCUUUAUGAGAU,

SEQ. ID NO. 2391: UUUAUGAGAUGCUGAGUGA)

could be introduced into cells at increasingly limiting concentrationsto determine whether any of the duplexes are hyperfunctional. Similarly,rationally designed sequences directed against

HIF1 Alpha

(SEQ. ID NO. 2392: GAAGGAACCUGAUGCUUUA,

SEQ. ID NO. 2393: GCAUAUAUCUAGAAGGUAU,

SEQ. ID NO. 2394: GAACAAAUACAUGGGAUUA,

SEQ. ID NO. 2395: GGACACAGAUUUAGACUUG), or

VEGF

(SEQ. ID NO. 2396: GAACGUACUUGCAGAUGUG,

SEQ. ID NO. 2397: GAGAAAGCAUUUGUUUGUA,

SEQ. ID NO. 2398: GGAGAAAGCAUUUGUUUGU,

SEQ. ID NO. 2399: CGAGGCAGCUUGAGUUAAA) could be introduced into cells atincreasingly limiting concentrations and screened for hyperfunctionalduplexes.

Example XVII Gene Silencing: Prophetic Example

Below is an example of how one might transfect a cell.

a. Select a cell line. The selection of a cell line is usuallydetermined by the desired application. The most important feature toRNAi is the level of expression of the gene of interest. It is highlyrecommended to use cell lines for which siRNA transfection conditionshave been specified and validated.

b. Plate the cells. Approximately 24 hours prior to transfection, platethe cells at the appropriate density so that they will be approximately70-90% confluent, or approximately 1×10⁵ cells/ml at the time oftransfection. Cell densities that are too low may lead to toxicity dueto excess exposure and uptake of transfection reagent-siRNA complexes.Cell densities that are too high may lead to low transfectionefficiencies and little or no silencing. Incubate the cells overnight.Standard incubation conditions for mammalian cells are 37° C. in 5% CO₂.Other cell types, such as insect cells, require different temperaturesand CO₂ concentrations that are readily ascertainable by persons skilledin the art. Use conditions appropriate for the cell type of interest.

c. siRNA re-suspension. Add 20 μl siRNA universal buffer to each siRNAto generate a final concentration of 50 μM.

d. SiRNA-lipid complex formation. Use RNase-free solutions and tubes.Using the following table, Table XI: TABLE XI 96-well 24-well Mixture 1(TransIT-TKO-Plasmid dilution mixture) Opti-MEM 9.3 μl 46.5 μlTransIT-TKO (1 μg/μl) 0.5 μl 2.5 μl Mixture 1 Final Volume 10.0 μl 50.0μl Mixture 2 (siRNA dilution mixture) Opti-MEM 9.0 μl 45.0 μl siRNA (1μM) 1.0 μl 5.0 μl Mixture 2 Final Volume 10.0 μl 50.0 μl Mixture 3(siRNA-Transfection reagent mixture) Mixture 1 10 μl 50 μl Mixture 2 10μl 50 μl Mixture 3 Final Volume 20 μl 100 μl Incubate 20 minutes at roomtemperature. Mixture 4 (Media-siRNA/Transfection reagent mixture)Mixture 3 20 μl 100 μl Complete media 80 μl 400 μl Mixture 4 FinalVolume 100 μl 500 μlIncubate 48 hours at 37° C.Transfection. Create a Mixture 1 by combining the specified amounts ofOPTI-MEM serum free media and transfection reagent in a sterilepolystyrene tube. Create a Mixture 2 by combining specified amounts ofeach siRNA with OPTI-MEM media in sterile 1 ml tubes. Create a Mixture 3by combining specified amounts of Mixture 1 and Mixture 2. Mix gently(do not vortex) and incubate at room temperature for 20 minutes. Createa Mixture 4 by combining specified amounts of Mixture 3 to completemedia. Add appropriate volume to each cell culture well. Incubate cellswith transfection reagent mixture for 24-72 hours at 37° C. Thisincubation time is flexible. The ratio of silencing will remainconsistent at any point in the time period. Assay for gene silencingusing an appropriate detection method such as RT-PCR, Western blotanalysis, immunohistochemistry, phenotypic analysis, mass spectrometry,fluorescence, radioactive decay, or any other method that is now knownor that comes to be known to persons skilled in the art and that fromreading this disclosure would useful with the present invention. Theoptimal window for observing a knockdown phenotype is related to themRNA turnover of the gene of interest, although 24-72 hours is standard.Final Volume reflects amount needed in each well for the desired cellculture format. When adjusting volumes for a Stock Mix, an additional10% should be used to accommodate variability in pipetting, etc.Duplicate or triplicate assays should be carried out when possible.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departure from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. A kit for gene silencing, wherein said kit is comprised of a pool of at least two siRNA duplexes, each of which is comprised of a sequence that is complementary to a portion of the sequence of one or more target messenger RNA, and each of which is selected using selection criteria that are embodied in a formula comprising: selection criteria are embodied in a formula comprising: (−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(number of A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula X wherein position numbering begins at the 5′-most position of a sense strand, and A₁=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; A₂=1 if A is the base at position 2 of the sense strand, otherwise its value is 0; A₃=1 if A is the base at position 3 of the sense strand, otherwise its value is 0; A₄=1 if A is the base at position 4 of the sense strand, otherwise its value is 0; A₅=1 if A is the base at position 5 of the sense strand, otherwise its value is 0; A₆=1 if A is the base at position 6 of the sense strand, otherwise its value is 0; A₇=1 if A is the base at position 7 of the sense strand, otherwise its value is 0; A₁₀=1 if A is the base at position 10 of the sense strand, otherwise its value is 0; A₁₁=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; A₁₃=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; A₁₉=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; C₃=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; C₄=1 if C is the base at position 4 of the sense strand, otherwise its value is 0; C₅=1 if C is the base at position 5 of the sense strand, otherwise its value is 0; C₆=1 if C is the base at position 6 of the sense strand, otherwise its value is 0; C₇=1 if C is the base at position 7 of the sense strand, otherwise its value is 0; C₉=1 if C is the base at position 9 of the sense strand, otherwise its value is 0; C₁₇=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; C₁₈=1 if C is the base at position 18 of the sense strand, otherwise its value is 0; C₁₉=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; G₁=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; G₂=1 if G is the base at position 2 of the sense strand, otherwise its value is 0; G₈=1 if G is the base at position 8 on the sense strand, otherwise its value is 0; G₁₀=1 if G is the base at position 10 on the sense strand, otherwise its value is 0; G₁₃=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; G₁₉=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; U₁=1 if U is the base at position 1 on the sense strand, otherwise its value is 0; U₂=1 if U is the base at position 2 on the sense strand, otherwise its value is 0; U₃=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; U₄=1 if U is the base at position 4 on the sense strand, otherwise its value is 0; U₇=1 if U is the base at position 7 on the sense strand, otherwise its value is 0; U₉=1 if U is the base at position 9 on the sense strand, otherwise its value is 0; U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0; U₁₅=1 if U is the base at position 15 on the sense strand, otherwise its value is 0; U₁₆=1 if U is the base at position 16 on the sense strand, otherwise its value is 0; U₁₇=1 if U is the base at position 17 on the sense strand, otherwise its value is 0; U₁₈=1 if U is the base at position 18 on the sense strand, otherwise its value is
 0. 2. A method for selecting an siRNA, said method comprising: applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs; and determining the relative functionality of the at least two siRNAs, wherein said section criteria are non-target specific criteria, said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene, and said selection criteria are embodied in a formula comprising: (−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(number of A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula X wherein position numbering begins at the 5′-most position of a sense strand, and A₁=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; A₂=1 if A is the base at position 2 of the sense strand, otherwise its value is 0; A₃=1 if A is the base at position 3 of the sense strand, otherwise its value is 0; A₄=1 if A is the base at position 4 of the sense strand, otherwise its value is 0; A₅=1 if A is the base at position 5 of the sense strand, otherwise its value is 0; A₆=1 if A is the base at position 6 of the sense strand, otherwise its value is 0; A₇=1 if A is the base at position 7 of the sense strand, otherwise its value is 0; A₁₀=1 if A is the base at position 10 of the sense strand, otherwise its value is 0; A₁₁=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; A₁₃=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; A₁₉=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; C₃=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; C₄=1 if C is the base at position 4 of the sense strand, otherwise its value is 0; C₅=1 if C is the base at position 5 of the sense strand, otherwise its value is 0; C₆=1 if C is the base at position 6 of the sense strand, otherwise its value is 0; C₇=1 if C is the base at position 7 of the sense strand, otherwise its value is 0; C₉=1 if C is the base at position 9 of the sense strand, otherwise its value is 0; C₁₇=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; C₁₈=1 if C is the base at position 18 of the sense strand, otherwise its value is 0; C₁₉=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; G₁=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; G₂=1 if G is the base at position 2 of the sense strand, otherwise its value is 0; G₈=1 if G is the base at position 8 on the sense strand, otherwise its value is 0; G₁₀=1 if G is the base at position 10 on the sense strand, otherwise its value is 0; G₁₃=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; G₁₉=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; U₁=1 if U is the base at position 1 on the sense strand, otherwise its value is 0; U₂=1 if U is the base at position 2 on the sense strand, otherwise its value is 0; U₃=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; U₄=1 if U is the base at position 4 on the sense strand, otherwise its value is 0; U₇=1 if U is the base at position 7 on the sense strand, otherwise its value is 0; U₉=1 if U is the base at position 9 on the sense strand, otherwise its value is 0; U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0; U₁₅=1 if U is the base at position 15 on the sense strand, otherwise its value is 0; U₁₆=1 if U is the base at position 16 on the sense strand, otherwise its value is 0; U₁₇=1 if U is the base at position 17 on the sense strand, otherwise its value is 0; U₁₈=1 if U is the base at position 18 on the sense strand, otherwise its value is
 0. 3. A method according to claim 1, further comprising comparing the internal stability profiles of said at least two siRNAs.
 4. A method according to claim 2, further comprising comparing the internal stability profiles of said at least two siRNAs.
 5. A method according to claim 1, further comprising selecting either for or against sequences that contain motifs that induce cellular stress.
 6. A method according to claim 2, further comprising selecting either for or against sequences that contain motifs that induce cellular stress.
 7. A method according to claim 1, further comprising selecting either for or against sequences that comprise stability motifs.
 8. A method according to claim 2, further comprising selecting either for or against sequences that comprise stability motifs.
 9. A method of gene silencing, comprising introducing into a cell at least one siRNA selected according to a method of claim
 1. 10. A method of gene silencing, comprising introducing into a cell at least one siRNA selected according to a method of claim
 2. 11. A method according to claim 1, wherein said introducing is by allowing passive uptake of the at least one siRNA.
 12. A method according to claim 2, wherein said introducing is by allowing passive uptake of the at least one siRNA.
 13. A method according claim 9, wherein said introducing in through the use of a vector.
 14. A method for developing an siRNA algorithm for selecting siRNA, said method comprising: (a) selecting a set of siRNA; (b) measuring gene silencing ability of each siRNA from said set; (c) determining relative functionality of each siRNA; (d) determining improved functionality based on the following variables: the presence or absence of a particular nucleotide at a particular position, the total number of As and Us in positions 15-19, the number of times that the same nucleotide repeats within a given sequence, and the total number of Gs and Cs; and (e) developing an algorithm using the information of step (d).
 15. A method of selecting an siRNA with improved functionality, said method comprising using the algorithm of claim
 14. 16. A kit, wherein said kit is comprised of at least two siRNAs, wherein said at least two siRNAs comprise a first optimized siRNA and a second optimized siRNA, wherein said first optimized siRNA and said second optimized siRNA are optimized according a formula comprising: (−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(number of A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula X wherein position numbering begins at the 5′-most position of a sense strand, and A₁=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; A₂=1 if A is the base at position 2 of the sense strand, otherwise its value is 0; A₃=1 if A is the base at position 3 of the sense strand, otherwise its value is 0; A₄=1 if A is the base at position 4 of the sense strand, otherwise its value is 0; A₅=1 if A is the base at position 5 of the sense strand, otherwise its value is 0; A₆=1 if A is the base at position 6 of the sense strand, otherwise its value is 0; A₇=1 if A is the base at position 7 of the sense strand, otherwise its value is 0; A₁₀=1 if A is the base at position 10 of the sense strand, otherwise its value is 0; A₁₁=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; A₁₃=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; A₁₉=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; C₃=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; C₄=1 if C is the base at position 4 of the sense strand, otherwise its value is 0; C₅=1 if C is the base at position 5 of the sense strand, otherwise its value is 0; C₆=1 if C is the base at position 6 of the sense strand, otherwise its value is 0; C₇=1 if C is the base at position 7 of the sense strand, otherwise its value is 0; C₉=1 if C is the base at position 9 of the sense strand, otherwise its value is 0; C₁₇=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; C₁₈=1 if C is the base at position 18 of the sense strand, otherwise its value is 0; C₁₉=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; G₁=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; G₂=1 if G is the base at position 2 of the sense strand, otherwise its value is 0; G₈=1 if G is the base at position 8 on the sense strand, otherwise its value is 0; G₁₀=1 if G is the base at position 10 on the sense strand, otherwise its value is 0; G₁₃=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; G₁₉=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; U₁=1 if U is the base at position 1 on the sense strand, otherwise its value is 0; U₂=1 if U is the base at position 2 on the sense strand, otherwise its value is 0; U₃=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; U₄=1 if U is the base at position 4 on the sense strand, otherwise its value is 0; U₇=1 if U is the base at position 7 on the sense strand, otherwise its value is 0; U₉=1 if U is the base at position 9 on the sense strand, otherwise its value is 0; U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0; U₁₅=1 if U is the base at position 15 on the sense strand, otherwise its value is 0; U₁₆=1 if U is the base at position 16 on the sense strand, otherwise its value is 0; U₁₇=1 if U is the base at position 17 on the sense strand, otherwise its value is 0; U₁₈=1 if U is the base at position 18 on the sense strand, otherwise its value is
 0. 17. A method for identifying hyperfunctional siRNA, comprising: applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; and determining the relative functionality of the at least two siRNAs and assigning each of the at least two siRNAs a functionality score; and selecting siRNAs from the at least two siRNAs that have a functionality score that reflects greater than 80 percent silencing at a concentration in the picomolar range, wherein said greater than 80 percent silencing endures for greater than 120 hours.
 18. A method according to claim 1, wherein said siRNA are unimolecular.
 19. A method according to claim 2, wherein said siRNA are unimolecular.
 20. A method according to claim 14, wherein said siRNA are unimolecular.
 21. A method according to claim 16, wherein said siRNA are unimolecular.
 22. A method according to claim 17, wherein said siRNA are unimolecular.
 23. A method according to claim 1, wherein said siRNA are comprised of two separate polynucleotide strands.
 24. A method according to claim 2, wherein said siRNA are comprised of two separate polynucleotide strands.
 25. A method according to claim 14, wherein said siRNA are comprised of two separate polynucleotide strands.
 26. A method according to claim 16, wherein said siRNA are comprised of two separate polynucleotide strands.
 27. A method according to claim 17, wherein said siRNA are comprised of two separate polynucleotide strands.
 28. A method according to claim 1, wherein said siRNA are expressed from one or more vectors.
 29. A method according to claim 2, wherein said siRNA are expressed from one or more vectors.
 30. A method according to claim 14, wherein said siRNA are expressed from one or more vectors.
 31. A method according to claim 16, wherein said siRNA are expressed from one or more vectors.
 32. A method according to claim 17, wherein said siRNA are expressed from one or more vectors.
 33. A method according to claim 1, wherein two or more genes are silenced by a single administration of siRNA.
 34. A method according to claim 2, wherein two or more genes are silenced by a single administration of siRNA.
 35. A method according to claim 14, wherein two or more genes are silenced by a single administration of siRNA.
 36. A method according to claim 16, wherein two or more genes are silenced by a single administration of siRNA.
 37. A method according to claim 17, wherein two or more genes are silenced by a single administration of siRNA.
 38. A kit according to claim 13, wherein one or more of said siRNA are unimolecular.
 39. A kit according to claim 13, wherein one or more of said siRNA are comprised of two separate polynucleotide strands.
 40. A kit according to claim 13, wherein one or more of said siRNA are capable of silencing the Bcl2 gene.
 41. A method for developing an siRNA algorithm for selecting functional and hyperfunctional siRNAs for a given sequence, comprising: (a) selecting a set of siRNAs; (b) measuring the gene silencing ability of each siRNA from said set; (c) determining the relative functionality of each siRNA; (d) determining the amount of improved functionality based on the following variables: the total GC content, melting temperature of the siRNA, GC content at positions 15-19, the presence or absence of a particular nucleotide at a particular position, relative thermodynamic stability at particular positions in a duplex, and the number of times that the same nucleotide repeats within a given sequence; and (e) developing an algorithm using the information of step (d). 